U.S. patent application number 12/134390 was filed with the patent office on 2009-05-07 for identification of trpml3 (mcoln3) as a salty taste receptor and use in assays for identifying taste (salty) modulators and/or therapeutics that modulate sodium transport, absorption or excretion and/or aldosterone and/or vasopressin production or release.
Invention is credited to Paul Brust, Na Gao, Peter Hevezi, Dalia Kalabat, Min Lu, Bryan Moyer, Guy Servant, Hortensia Soto, Evan Carl White, Mark Williams, Albert Zlotnik.
Application Number | 20090117563 12/134390 |
Document ID | / |
Family ID | 41328825 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090117563 |
Kind Code |
A1 |
Moyer; Bryan ; et
al. |
May 7, 2009 |
Identification of TRPML3 (MCOLN3) as a Salty Taste Receptor and Use
in Assays for Identifying Taste (Salty) Modulators and/or
Therapeutics that Modulate Sodium Transport, Absorption or
Excretion and/or Aldosterone and/or Vasopressin Production or
Release
Abstract
This invention relates to the elucidation that TRPML3 is
involved in salty taste perception in primates including humans and
likely other mammals (given the significance of sodium and other
ions to physiological functions and conditions this phenotype is
likely strongly conserved in different animals). The invention also
relates to the discovery that the TRPML3 gene also modulates one or
more of sodium metabolism, sodium excretion, blood pressure, fluid
retention, cardiac function and urinary functions such as urine
production and excretion. The invention also relates to transgenic
animals that have been engineered to express or knock out TRPML3
expression and assays using TRPML3 expressing animals, cells and
isolated ion channel polypeptides for identifying compounds that
modulate TRPML3-associated functions including salty taste, sodium
metabolism, sodium excretion, blood pressure, fluid retention,
cardiac function and urinary functions such as urine production and
excretion.
Inventors: |
Moyer; Bryan; (San Diego,
CA) ; Zlotnik; Albert; (San Diego, CA) ;
Hevezi; Peter; (Encinitas, CA) ; Soto; Hortensia;
(San Diego, CA) ; Kalabat; Dalia; (El Cajon,
CA) ; Lu; Min; (San Diego, CA) ; Gao; Na;
(San Diego, CA) ; White; Evan Carl; (Fair Oaks,
CA) ; Servant; Guy; (San Diego, CA) ; Brust;
Paul; (San Diego, CA) ; Williams; Mark;
(Carlsbad, CA) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP;INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W., SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Family ID: |
41328825 |
Appl. No.: |
12/134390 |
Filed: |
June 6, 2008 |
Related U.S. Patent Documents
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Application
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Patent Number |
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60929017 |
Jun 8, 2007 |
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60929007 |
Jun 8, 2007 |
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60947052 |
Jun 29, 2007 |
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60935297 |
Aug 3, 2007 |
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60987611 |
Nov 13, 2007 |
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60988938 |
Nov 19, 2007 |
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60991274 |
Nov 30, 2007 |
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60991289 |
Nov 30, 2007 |
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60992502 |
Dec 5, 2007 |
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60992517 |
Dec 5, 2007 |
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61017244 |
Dec 28, 2007 |
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61043257 |
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61053310 |
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Current U.S.
Class: |
435/6.13 ;
435/252.3; 435/254.2; 435/29; 435/325; 435/348; 435/349; 435/350;
435/351; 435/353; 435/354; 435/358; 435/363; 435/365; 435/366;
435/7.1; 530/350 |
Current CPC
Class: |
G01N 33/566 20130101;
C40B 40/10 20130101; G01N 33/5044 20130101; C12Q 2600/158 20130101;
C12Q 1/6883 20130101; G01N 2500/10 20130101; C12Q 2600/136
20130101; G01N 2333/726 20130101; C40B 30/04 20130101; G01N 33/6872
20130101 |
Class at
Publication: |
435/6 ; 435/325;
530/350; 435/348; 435/349; 435/252.3; 435/254.2; 435/365; 435/358;
435/366; 435/353; 435/354; 435/350; 435/351; 435/363; 435/29;
435/7.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 5/00 20060101 C12N005/00; C07K 14/705 20060101
C07K014/705; C12N 5/06 20060101 C12N005/06; G01N 33/53 20060101
G01N033/53; C12N 1/21 20060101 C12N001/21; C12N 1/19 20060101
C12N001/19; C12Q 1/02 20060101 C12Q001/02 |
Claims
1. Isolated taste, adrenal, pituitary, parathyroid, melanocyte, or
urinary organ cells or an enriched taste cell sample wherein said
isolated or enriched cell sample comprises cells that express a
TRPML3 ion channel polypeptide.
2. (canceled)
3. (canceled)
4. (canceled)
5. The isolated cells or enriched cell sample of claim 1 wherein
said TRPML3 ion channel polypeptide possesses at least 90% sequence
identity to a TRPML3 polypeptide selected from the polypeptides
having SEQ ID NO: 2, 4, 10, 12, 14, 16, 22, 24, 26, 28, 30, 32 and
34 or the TRPML3 polypeptide encoded by SEQ ID NO:39 or 40.
6. The isolated cells or enriched cell sample of claim 5 wherein
said TRPML3 ion channel polypeptide possesses at least 95% sequence
identity to a TRPML3 polypeptide selected from the polypeptides
having SEQ ID NO: 2, 4, 10, 12, 14, 16, 22, 24, 26, 28, 30, 32 and
34 or the TRPML3 polypeptide encoded by SEQ ID NO:39 or 40.
7. The isolated cells or enriched cell sample of claim 5 wherein
said TRPML3 ion channel polypeptide has the sequence contained in
SEQ ID NO:2, 4 or 10 or the TRPML3 polypeptide encoded by SEQ ID
NO:39 or 40.
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. The isolated cells or cell sample of claim 1 which comprises
taste cells which respond to salty taste.
14. The isolated cells or cell sample of claim 13 wherein the taste
cells are selected from human, non-human primate, rodent, canine or
feline taste cells.
15. The isolated cells or cell sample of claim 14 which are human
or non-human primate taste cells.
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. The isolated taste cell or enriched cell sample of claim 1
wherein the taste cell is a human salty taste cell.
25. (canceled)
26. (canceled)
27. (canceled)
28. The isolated cell or enriched cell sample of claim 1 which
further expresses NALCN, NKAIN3, TRPML1 and/or TRPML2.
29. An isolated taste receptor that modulates salty taste
perception comprising a TRPML3 polypeptide or variant thereof that
modulates salty taste in mammals.
30. (canceled)
31. (canceled)
32. (canceled)
33. The isolated taste receptor of claim 29 which comprises a
mammalian, avian, amphibian, fish, or reptilian TRPML3 ion
channel.
34. The isolated taste receptor of claim 29 which is mammalian or a
chicken, zebrafish or Xenopus taste receptor.
35. The isolated taste receptor of claim 29 which comprises a
TRPML3 selected from human, murine, rat, canine, feline, guinea
pig, pig, horse, cow, goat, sheep, bear, monkey, gorilla,
chimpanzee, orangutan, cynomolgus monkey, gibbon, gazelle, macaque,
and zebra TRPML3.
36. The isolated taste receptor of claim 29 wherein said TRPML3 ion
channel polypeptide possesses at least 90% sequence identity to a
TRPML3 polypeptide selected from the polypeptides having SEQ ID NO:
2, 4, 10, 12, 14, 16, 22, 24, 26, 28, 30, 32 and 34 or the TRPML3
polypeptide encoded by SEQ ID NO:39 or 40.
37. The isolated taste receptor of claim 29 wherein said TRPML3 ion
channel polypeptide possesses at least 95% sequence identity to a
TRPML3 polypeptide selected from the polypeptides having SEQ ID NO:
2, 4, 10, 12, 14, 16, 22, 24, 26, 28, 30, 32 and 34 or the TRPML3
polypeptide encoded by SEQ ID NO:39 or 40.
38. The isolated taste receptor of claim 29 wherein the TRPML3
polypeptide expressed therein has a mutation that renders the ion
channel more or less sensitive to sodium.
39. The isolated taste receptor of claim 29 wherein the TRPML3
polypeptide expressed therein has a mutation that renders the ion
channel more or less permeable to sodium.
40. The isolated taste receptor of claim 29 wherein the TRPML3
polypeptide expressed therein has a mutation that maintains the ion
channel in an "open" or "closed" orientation.
41. The isolated taste receptor of claim 29 wherein the TRPML3
polypeptide expressed therein has a mutation that is toxic to cells
containing the ion channel.
42. The isolated taste receptor of claim 29 wherein the TRPML3
polypeptide expressed therein has a mutation such that when this
ion channel is expressed in a cell sodium or calcium influx and
efflux is uncontrolled.
43. The isolated taste receptor of claim 29 which comprises another
ion channel polypeptide.
44. The isolated taste receptor of claim 43 wherein said other ion
channel is NKAIN3, NALCN., TRPML1 or TRPML2.
45. The isolated taste receptor of claim 29 which is a human or
non-human primate salty taste receptor.
46. The isolated taste receptor of claim 45 which comprises a human
salty taste receptor.
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. (canceled)
54. (canceled)
55. (canceled)
56. (canceled)
57. (canceled)
58. (canceled)
59. (canceled)
60. (canceled)
61. (canceled)
62. (canceled)
63. (canceled)
64. (canceled)
65. (canceled)
66. (canceled)
67. (canceled)
68. (canceled)
69. (canceled)
70. (canceled)
71. (canceled)
72. (canceled)
73. A recombinant cell which expresses a salty taste receptor
comprising TRPML3 or a variant thereof.
74. The recombinant cell of claim 73 which is a yeast, amphibian,
insect, bacterial, reptile, avian, or mammalian cell.
75. The recombinant cell of claim 74 which is an amphibian oocyte
or mammalian cell.
76. The recombinant cell of claim 75 which is selected from a
HEK-293, COS, CHO, and a BHK cells.
77. The recombinant cell of claim 73 which transiently expresses
said TRPML3 polypeptide.
78. The recombinant cell of claim 73 which stably expresses said
TRPML3 polypeptide.
79. The recombinant cell of claim 73 which comprises a baculovirus
expression vector.
80. The recombinant cell of claim 79 wherein said vector is a
BacMam vector.
81. The recombinant cell sample of claim 73 wherein said TRPML3 is
selected from a mammalian, avian, amphibian, fish, and reptilian
TRPML3.
82. The recombinant cell of claim 73 which expresses a mammalian
TRPML3 or a chicken, zebrafish or Xenopus TRPML3.
83. The recombinant cell of claim 73 which expresses a mammalian
TRPML3 is selected from the group consisting of human, murine, rat,
canine, feline, guinea pig, pig, horse, cow, goat, sheep, bear,
monkey, gorilla, chimpanzee, orangutan, macaque, cynomolgus monkey,
gibbon, gazelle, and zebra TRPML3.
84. The recombinant cell of claim 73 wherein said TRPML3 ion
channel polypeptide possesses at least 90% sequence identity to a
TRPML3 polypeptide selected from the polypeptides having SEQ ID NO:
2, 4, 10, 12, 14, 16, 22, 24, 26, 28, 30, 32 and 34 or the TRPML3
polypeptide encoded by SEQ ID NO:39 or 40.
85. The recombinant cell of claim 73 wherein said TRPML3 ion
channel polypeptide possesses at least 95% sequence identity to a
TRPML3 polypeptide selected from the polypeptides having SEQ ID NO:
2, 4, 10, 12, 14, 16, 22, 24, 26, 28, 30, 32 and 34 or the TRPML3
polypeptide encoded by SEQ ID NO:39 or 40.
86. The recombinant cell of claim 73 wherein said TRPML3 ion
channel polypeptide has the sequence contained in SEQ ID NO:2 or 4
or comprises the TRPML3 polypeptide encoded by SEQ ID NO:39 or
40.
87. The recombinant cell of claim 73 wherein the TRPML3 polypeptide
expressed by said cells has a mutation that renders the ion channel
more or less sensitive to sodium.
88. The recombinant cell of claim 73 wherein the TRPML3 polypeptide
expressed by said cell has a mutation that renders the ion channel
more or less permeable to sodium.
89. The recombinant cell of claim 73 wherein the TRPML3 polypeptide
expressed by said cell has a mutation that maintains the ion
channel in an "open" orientation.
90. The recombinant cell of claim 73 wherein the TRPML3 polypeptide
expressed by said cell sample has a mutation that is toxic to some
cells.
91. The recombinant cell of claim 73 wherein the TRPML3 polypeptide
expressed by said cells or enriched cell sample has a mutation that
results in a cell wherein sodium influx and efflux is
uncontrolled.
92. The recombinant cell of claim 73 wherein said TRPML3 ion
channel responds to salty taste modulators.
93. The recombinant cell of claim 73 which expresses another sodium
ion channel polypeptide.
94. The recombinant cell of claim 89 wherein said other sodium
channel is NALCN, NKAIN3, TRPML1 or TRPML2.
95. An assay for identifying compounds that agonize, antagonize or
enhance an activity of TRPML3 comprising contacting a recombinant
cell according to claim 73 or a primary cell, acutely dissociated
cell, or other cell which endogenously expresses a TRPML3 ion
channel polypeptide with a putative TRPML3 enhancer, agonist or
antagonist and determining the effect thereof on TRPML3
activity.
96. The assay of claim 95 wherein the endogenous cell is selected
from an adrenal cortex cell, parathyroid cell, taste bud cell,
urinary organ cell, melanocyte, adrenal or parathyroid cell.
97. The assay of claim 95 wherein the cell is a primary, acutely
disassociated, or other endogenous cell that expresses TRPML3.
98. The assay of claim 97 wherein the cell is an adrenal cortex,
pituitary, parathyroid, taste, or melanocyte.
99. The assay of claim 98 wherein the cell expresses a rodent or
human or non-human primate TRPML3 sequence.
100. The assay of claim 98 wherein the TRPML3 is encoded by a
sequence having optimized codons favoring enhanced expression in
the recombinant host cell.
101. The assay of claim 98 wherein the TRPML3 has a sequence at
least 90% identical to a polypeptide selected from those contained
in SEQ ID NO:2, 4, 10, 12, 14, 16, 22, 24, 26, 28, 30, 32, and 34
or encoded by the polypeptide in SEQ ID NO:39 or 40.
102. The assay of claim 98 wherein the TRPML3 has a sequence at
least 95% identical to a polypeptide selected from those contained
in SEQ ID NO:2, 4, 10, 12, 14, 16, 22, 24, 26, 28, 30, 32, and 34
or encoded by the polypeptide in SEQ ID NO:39 or 40.
103. The assay of claim 98 wherein the TRPML3 is a wild-type human
sequence or a rodent or human sequence comprising the
Varitint-waddler mutation (A419P).
104. The assay of claim 98 wherein the TRPML3 is a human sequence
encoded by SEQ ID NO:1, 3, 17 or 18.
105. The assay of claim 98 which is an electrophysiological
assay.
106. The assay of claim 105 wherein the recombinant cell an
amphibian oocyte.
107. The assay of claim 105 wherein the recombinant cell is a
mammalian cell.
108. The assay of claim 105 wherein said assay is an
electrophysiological assay which uses an ion sensitive dye or
fluorophore.
109. The assay of claim 105 wherein said assay is a two electrode
voltage clamping assay.
110. The assay of claim 109 wherein the test cell is a Xenopus
oocyte.
111. The assay of claim 107 wherein said mammalian cell is selected
from the group consisting of a HEK293, HEK293T, Swiss3T3, CHO, BHK,
NIH3T3, and COS cell.
112. The assay of claim 105 wherein the cell is a Xenopus
oocyte.
113. The assay of claim 105 wherein said assay is a patch clamp
assay.
114. The assay of claim 105 which uses a membrane potential dye is
selected from the group consisting of Molecular Devices Membrane
Potential Kit (Cat#R8034), Di-4-ANEPPS (pyridinium,
4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)-1-(3-sulfopropyl)hydroxid-
e, inner salt, DiSBACC4(2)(bis-(1,2-dibabituric acid)-triethine
oxanol), Cc-2-DMPE (Pacific Blue
1,2-dietradecanoyl-sn-glycerol-3phosphoethanolamine,
triethylammonium salt) and SBFI-AM (1,3-benzenedicrboxylic acid,
4,4-[1,4,10-trioxa-7,13-diazacylopentadecane-7,13-diylbis(5-methoxy-6,1,2-
-benzofurandiyl)}bis-tetrakis{(acetyloxy)methyl}ester (Molecular
Probes).
115. The method of claim 105 which uses a sodium sensitive dye.
116. The method of claim 115 wherein the sodium sensitive dye is
sodium green tetraacetate (Molecular Probes) or Na-sensitive Dye
Kit (Molecular Devices).
117. The assay of claim 97 wherein the assay measures activity by
an ion flux assay.
118. The assay of claim 117 which uses atomic absorption
spectroscopy to detect ion flux.
119. The assay of claim 105 which uses a fluorescence plate reader
(FLIPR).
120. The assay of claim 105 which uses a voltage imaging plate
reader (VIPR).
121. The assay of claim 105 which uses an automated
electrophysiology instrument.
122. The assay of claim 121 which uses an IonWorks assay
system.
123. The assay of claim 105 which uses a membrane potential dye
selected from the group consisting of Molecular Devices Membrane
Potential Kit (cat#8034), Di-4-ANEPPS (pyridinium,
4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)-1-(3-sulfopropyl)-hydroxi-
de, inner salt); DiSBACC4(2)(bis-(1.2-dibarbituric acid)-trimethine
oxanol); DiSBAC4(3) (bis-(1,3-dibarbituric acid)-trimethine
oxanol); CC-2-DPME (Pacific Blue
1,2-dietradecanoyl-sn-glycerol-3-phosphoethanolamine,
triethylammonium salt) and SBFI-AM (1,3-Benzenedicarboxylic acid,
4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,1-
,2-benzofurandiyl)]bis-tetrakis[(acetyloxy)methyl]ester (Molecular
Probes).
124. The assay of claim 99 wherein the identified agonist,
antagonist, or enhancer compounds are evaluated in a taste
test.
125. The assay of claim 99 wherein the effect of the identified
agonist, antagonist or enhancer on aldosterone production is tested
in an animal.
126. The assay of claim 99 wherein the effect of the identified
agonist, antagonist or enhancer on vasopressin release is tested in
an animal.
127. The assay of claim 99 wherein the effect of said identified
antagonist, agonist or enhancer compound on at least one of
Addison's disease, hair loss, hair or fur discoloration, taste cell
regeneration, pituitary cell regeneration, adrenal cell
regeneration, melanocyte cell regeneration, blood pressure, fluid
retention, sodium metabolism and urine production is tested in an
animal.
128. The assay of claim 99 wherein the effect of the identified
compound for the treatment of wherein the disease or condition is
selected from edema, blood pressure (hyper or hypotension), liver
cirrhosis, primary hyperaldosterinia, renal dysfunction, diabetes
(Type I or II) and the pathological symptoms associated therewith
including circulatory problems, edema, ocular disorders relating to
poor circulation, hypercortisolaemia, atherosclerosis or obesity,
e.g., abdominal obesity, as well as liver disease, sexual
dysfunction (male or female), cerebrovascular disease, vascular
disease, retinopathy, neuropathy, insulinopathy, endothelial
dysfunction, baroreceptor dysfunction, migraine headaches, hot
flashes, and premenstrual tension and other cardiovascular
conditions such as atherosclerosis, heart failure, congestive heart
failure, vascular disease, stroke, myocardial infarction,
endothelial dysfunction, ventricular hypertrophy, renal
dysfunction, target-organ damage, thrombosis, cardiac arrhythmia,
plaque rupture and aneurysm or another condition treatable by an
aldosterone agonist or antagonist is evaluated in an appropriate in
vitro or in vivo animal model.
129. The assay of claim 99 wherein the effect of the identified
compound for the treatment of wherein the disease or condition is
selected from cystic kidney disease, acquired renal cystic disease,
ocular circulation related disorders such as myopia; nausea,
emesis, sexual dysfunction (male or female), edema, hypertension,
congestive heart failure (ranging from class II of the New York
Heart Association to florid pulmonary edema), periodic idiopathic
edema, nephrotic syndrome, ascites due to cirrhosis or other
causes, cerebral edema of various causes, dilutional hyponatremia
and metabolic alterations collectively known as the syndrome of
inappropriate ADH secretion and other diseases or conditions
wherein vasodilation and/or antioxytocic activity or the
administration of a vasopressin agonist or antagonist is
therapeutically desirable is tested in an appropriate in vitro or
in vivo model.
130. (canceled)
131. (canceled)
133. (canceled)
134. (canceled)
135. (canceled)
136. (canceled)
137. (canceled)
138. (canceled)
139. (canceled)
140. (canceled)
141. (canceled)
142. (canceled)
143. A method of using a probe specific to a TRPML3 gene or gene
product to identify and/or isolate and or enrich salty taste
specific cells in a sample.
144. (canceled)
145. (canceled)
146. (canceled)
147. (canceled)
148. (canceled)
150. (canceled)
151. (canceled)
152. (canceled)
153. (canceled)
154. (canceled)
155. A method of identifying putative salty taste modulators in a
binding assay comprising providing a TRPML3 polypeptide or cell
which expresses TRPML3 and contacting said polypeptide or cell with
putative TRPML3 modulatory compounds and identifying potential
TRPML3 modulators based on their specific binding to TRPML3
polypeptide.
156. The binding assay of claim 155 which is a direct binding
assay.
157. The binding assay of claim 155 which is a competitive binding
assay.
158. The binding assay of claim 155 which uses a mammalian TRPML3
polypeptide.
159. The binding assay of claim 158 which uses a TRPML3 polypeptide
that possesses at least 90% sequence identity to a TRPML3
polypeptide selected from those in SEQ ID NO:2, 4, 10, 12, 14, 16,
22, 24, 26, 28, 30, 31 and 34 or a polypeptide encoded by SEQ ID
NO:39 or 40.
160. The binding assay of claim 158 which uses a TRPML3 polypeptide
that possesses at least 95% sequence identity to a TRPML3
polypeptide selected from those in SEQ ID NO:2, 4, 10, 12, 14, 16,
22, 24, 26, 28, 30, 31 and 34 or a polypeptide encoded by SEQ ID
NO:39 or 40.
161. The binding assay of claim 160 which uses wild-type human,
non-human primate or rodent TRPML3 polypeptide or a TRPML3
polypeptide having a A419P mutation.
162. The binding assay of claim 158 which uses a radionuclide,
fluorophore or enzyme to facilitate detection of binding.
163. The binding assay of claim 158 wherein the ion channel is
expressed by a mammalian cell.
164. The binding assay of claim 158 wherein the TRPML3 polypeptide
is monomeric or polymeric.
165. The binding assay of claim 158 wherein the TRPML3 is
heteropolymeric and comprises TRPML1 or TRPML2 or the TRPML3 is
expressed in association with TRPML1 or TRPML2.
166. (canceled)
167. (canceled)
168. (canceled)
169. (canceled)
170. (canceled)
171. (canceled)
172. (canceled)
173. A method for identifying a modulator of TRPML3 utilizing a
mammalian cell or oocyte that expresses a functional TRPML3 sodium
channel with a putative TRPML3 modulatory compound, comprising: (i)
assaying the effect of said compound on sodium transport through
the TRPML3 channel; and (ii) identifying whether said compound is
an TRPML3 modulator based on its enhancing or inhibitory effect on
sodium transport.
174. The method of claim 173, further comprising (iii) confirming
that the compound identified modulates salty taste in human or
mammalian taste tests.
175. The method of claim 173 wherein the in vivo effect of the
identified compound on sodium excretion or urinary function is
tested in humans or mammals.
176. The method of claim 173, wherein the TRMPL3 is mammalian
TRMPL3.
177. The method of claim 164, wherein the TRMPL3 is human,
non-human primate, rodent, cow, pig, horse or sheep TRMPL3.
178. The method of claim 173, wherein said mammalian cell is
selected from the group consisting of a HEK293, HEK293T, Swiss3T3,
CHO, BHK, NIH3T3, and COS cells.
179. The method of claim 173 wherein the oocyte is a mammalian,
amphibian, avian or reptilian oocyte.
180. The method of claim 179, wherein said amphibian oocyte is a
Xenopus oocyte.
181. The method of claim 173, wherein said cell expresses an
additional gene or ion channel expressed in taste cells.
182. (canceled)
183. (canceled)
184. (canceled)
185. (canceled)
186. (canceled)
187. (canceled)
188. (canceled)
189. (canceled)
190. (canceled)
191. (canceled)
192. (canceled)
193. (canceled)
194. (canceled)
195. (canceled)
196. (canceled)
197. (canceled)
198. (canceled)
199. (canceled)
200. (canceled)
201. A mammalian or frog oocyte cell-based high-throughput assay
for the profiling and screening of putative modulators of TRPML3
comprising: contacting a test cell expressing TRPML3 or a variant,
fragment or functional equivalent and preloaded with a membrane
potential fluorescent dye or a sodium fluorescent dye with at least
one TRPML3 putative modulator compound in the presence of sodium or
lithium; and monitoring cation mediated changes in fluorescence of
the test cell in the presence of the putative modulator/TRPML3
interactions compared to changes in the absence of the modulator to
determine the extent of TRPML3 modulation.
202. The assay method of claim 201 in which the cation is
sodium.
203. The assay method of claim 201 in which the cation is
lithium.
204. The assay of claim 201 wherein the cation is potassium.
205. The assay method of claim 201 in which the test cell is
selected from the group consisting of MDCK, HEK293, HEK293 T, BHK,
COS, NIH3T3, U2OS, Swiss3T3 and CHO.
206. The assay method of claim 205 in which the test cell is a
HEK293 cell.
207. The assay method of claim 201 in which the method is used to
identify a compound as one which particularly modulates taste based
on a detectable change in fluorescence.
208. The assay method of claim 207 wherein the taste is salty
taste.
209. The assay method of claim 201 in which the test cells are
seeded onto a well of a multi-well test plate.
210. The assay method of claim 209 wherein the test cells are
contacted with a putative modulator by adding the putative
modulator to the well of the multi-well test plate.
211. The assay method of claim 210 wherein the test cells are
loaded with a membrane potential dye that allows for changes in
fluorescence to be detected.
212. The assay method of claim 201 wherein TRPML3 is a human TRPML3
that is encoded by human TRPML3 DNA sequences cloned from human
taste cell cDNA.
213. The assay method of claim 201 wherein the TRPML3 DNA is a
codon optimized or wild-type or mutant TRPML3 DNA.
214. The assay method of claim 213 wherein the TRPML3 DNA is
selected from those contained in SEQ ID NO: 1, 3, 17, 18, 39 and
40.
215. The assay of claim 201 wherein a fluorescence plate reader is
used to monitor changes in fluorescence.
216. The assay of claim 201 wherein a voltage imaging plate reader
is used to monitor changes in fluorescence.
217. The assay of claim 201 wherein the membrane potential dye is
selected from the group consisting of Di-4-ANEPPS (Pyridinium,
4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)-1-(3-sulfopropyl))-,
hydroxide, inner salt), DiSBACC4(2) (bis-(1,2-dibarbituric
acid)-trimethine oxanol), DiSBAC4(3) (bis-(1,3-dibarbituric
acid)-trimethine oxanol), CC-2-DMPE
(1,2-dietradecanoyl-sn-glycerol-3-phosphoethanolamine,
triethylammonium salt) and SBFI-AM (1,3-Benzenedicarboxylic acid,
4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,1-
-2-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl]ester.
218. A method for monitoring the activity of TRPML3 comprising:
providing a test cell transfected with a functional TRPML3 splice
variants and fragments thereof; seeding the test cell in the well
of a multi-well plate; dye-loading the seeded test cell with a
membrane potential dye in the well of the multi-well plate;
contacting the dye-loaded test cell with at least one putative
modulating compound and sodium in the well of the multi-well plate;
and monitoring any changes in fluorescence of the membrane
potential dye due to modulator/TRPML3 interactions using a
fluorescence plate reader or voltage intensity plate reader.
219. The method of claim 218 wherein the test cell is a HEK293
cell.
220. The method of claim 219 wherein the test cell is a HEK293T
cell.
221. The method of claim 218, wherein the membrane potential dye is
selected from the group consisting of Di-4-ANEPPS (Pyridinium,
4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-,
hydroxide, inner salt), DiSBAC4(2) (bis-(1,2-dibarbituric
acid)-trimethine oxanol), DiSBAC4(3) (bis-(1,3-dibarbituric
acid)-trimethine oxanol), CC-2-DMPE
(1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine,
triethylammonium salt) on SBFI-AM (1,3-Benzenedicarboxylic acid,
4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,1-
-2-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl]ester.
222. The method of claim 218, wherein the TRPML3 is a human TRPML3
cloned from human taste cell cDNA.
223. The method of claim 218 wherein the TRPML3 is selected from
the group consisting of: a naturally occurring human TRPML3, an
alternatively spliced human TRPML3, and a functional variant
thereof.
224. The method of claim 218 wherein the test cell is selected from
the group consisting of MDCK, HEK293, HEK293T, COS, BHK, NIH3T3,
Swiss3T3, U2OS and CHO cell.
225. A method for identifying a salty taste modulating compound
comprising: providing a test cell transfected or transformed with a
functional human TRPML3, splice variant, chimera or fragment
thereof; seeding the test cell in the well of a multi-well plate;
dye-loading the seeded test cell with a membrane potential dye in
the well of the multi-well plate; contacting the dye-loaded test
cell with at least one putative modulatory compound and sodium in
the well of the multi-well plate; monitoring any changes in
fluorescence of the membrane potential dye due to modulator/TRPML3
interactions using a fluorescence plate reader or voltage intensity
plate reader; and identifying at least one putative modulator as a
salty taste modulating compound based on the monitored changes in
fluorescence.
226. The method of claim 225 further comprising evaluating the
identified TRPML3 modulatory compound for effects on salty taste
perception.
227. The method of claim 212 wherein the test cell is selected from
the group consisting of MDCK, HEK293, HEK293T, COS, BHK, NIH3T3,
Swiss3T3, U2OS, and CHO.
228. The method of claim 227 wherein the test cell is an HEK293
cell.
229. The method of claim 228 wherein the test cell is a HEK293T
cell.
230. The method of claim 225 in which the method is used to
identify a compound as one which particularly modulates taste based
on a detectable change in fluorescence.
231. The method of claim 225 wherein the taste is salty taste.
232. The method of claim 225 wherein the test cells are contacted
with a putative modulator by adding the putative modulator to the
well of the multi-well test plate.
233. The method of claim 225 wherein the test cells are loaded with
a membrane potential dye that allows for changes in fluorescence to
be detected.
234. The method of claim 225 wherein the test cell expresses TRPML3
or a fragment or variant thereof.
235. The assay of claim 225, wherein the TRPML3 is selected from
the group consisting of: a naturally occurring human TRPML3, an
alternatively spliced human TRPML3, or a functional variant
thereof.
236. The assay of claim 225 wherein a fluorescence plate reader is
used to monitor changes in fluorescence.
237. The assay of claim 225 wherein a voltage imaging plate reader
is used to monitor changes in fluorescence.
238. The assay of claim 233 wherein the membrane potential dye is
selected from the group consisting of Di-4-ANEPPS (Pyridinium,
4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)-1-(3-sulfopropyl))-,
hydroxide, inner salt), DiSBACC4(2) (bis-(1,2-dibarbituric
acid)-trimethine oxanol), DiSBAC4(3) (bis-(1,3-dibarbituric
acid)-trimethine oxanol), CC-2-DMPE
(1,2-dietradecanoyl-sn-glycerol-3-phosphoethanolamine,
triethylammonium salt) and SBFI-AM (1,3-Benzene-dicarboxylic acid,
4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,1-
-2-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl]ester.
239. A method of identifying TRPML3 modulators by the use of a high
throughput patch clamp electrophysiological assay system.
240. The method of claim 239 which comprises an IonWorks automated
patch clamp system.
241. The method of claim 239 which uses a mammalian cell that
expresses a TRPML3 DNA.
242. The method of claim 241 wherein the mammalian cell is a CHO-K1
cell.
243. The method of claim 239 which is used to identify compounds
that open (activate) the TRPML3 ion channel.
244. The method of claim 226 which is used to identify compounds
that close (block) the TRPML3 ion channel.
245. The method of claim 243 wherein said compound is tested as a
salty taste enhancer.
246. The method of claim 243 wherein said compound is tested as a
salty taste blocker.
247. The method of claim 239 wherein the assay is run in the
standard mode where each well corresponds to a single cell.
248. The method of claim 239 wherein the assay is run in the
population patch clamp (PPC) mode where each well gives the average
current of 64 cells potentially increasing the overall success rate
("positive hits") and reducing well to well variability.
249. The method of claim 239 which uses a 384-well format
comprising TRPML3 expressing mammalian test cells.
250. The method of claim 239 which uses transiently transfected
CHO-K1 cells.
251. The method of claim 239 which uses stably transfected CHO-K1
cells.
252. The method of claim 239 which introduces the TRPML3 gene into
CHO-K1 cells by use of a BacMam expression system.
253. The method of claim 239 wherein the mammalian cells express
wild-type human TRPML3 DNA, a codon (human) optimized TRPML3 DNA
sequence or a TRPML3 mutant comprising a modification resulting in
a A419P mutation.
254. The method of claim 226 wherein TRPML3 function is measured
TRPML3 function measured using the perforated patch clamp technique
on an IonWorks Quattro instrument (MDS Analytical
Technologies).
255. (canceled)
256. (canceled)
257. (canceled)
258. (canceled)
259. (canceled)
Description
RELATED PROVISIONAL AND UTILITY APPLICATIONS
[0001] This application relates to earlier filed provisional
applications by the present Assignee Senomyx Inc relating to a
novel rationale for identifying primate taste specific genes and in
particular for identification of the primate salt receptor gene or
genes. These provisional applications U.S. Application Ser. No.
60/929,017, filed Jun. 8, 2007; U.S. Application Ser. No.
60/929,007, filed Jun. 8, 2007; U.S. Application Ser. No.
60/947,052, filed Jun. 29, 2007; U.S. Application Ser. No.
60/935,297, filed Aug. 3, 2007; U.S. Application Ser. No.
60/987,611, filed Nov. 13, 2007; U.S. Application Ser. No.
60/988,938, filed Nov. 19, 2007; U.S. Application Ser. No.
60/991,274, filed Nov. 30, 2007; U.S. Application Ser. No.
60/991,289, filed Nov. 30, 2007; U.S. Application Ser. No.
60/992,502, filed Dec. 5, 2007; U.S. Application Ser. No.
60/992,517, filed Dec. 5, 2007; U.S. Application Ser. No.
61/017,244, filed Dec. 28, 2007; U.S. Application Ser. No.
61/021,437, filed Jan. 16, 2008; U.S. Application Ser. No.
61/043,257, filed Apr. 8, 2008; and U.S. Application Ser. No.
61/053,310, filed May 10, 2008. In addition, this application
relates to, and claims priority to U.S. Ser. No. 11/808,356, filed
on Jun. 8, 2007, and to Attorney Docket No. 67824.703201 filed on
even date. All of the afore-mentioned provisional and
non-provisional applications are incorporated by reference in their
entireties herein.
[0002] These applications include disclosure relating to various
taste specific genes including TRPML3 or MCOLN3 as this gene is
alternatively known. This gene was highlighted as a gene to be
functionalized by the inventors in electrophysiological assays.
This gene was theorized by the inventors to be a candidate gene
encoding a salty taste receptor because it was predicted to encode
a taste specific sodium ion channel regulating salty taste in
primates (e.g., humans), rodents and other animals. As described
herein, in vitro and in vivo (animal) assays using cells (mammalian
and amphibian cells) and rodents expressing wild type or mutated
TRPML3 polypeptides have confirmed that TRPML3 or MCOLN3 is
involved in salty taste perception as well as affecting other
biological functions unrelated to taste such as hearing and balance
(as evidenced by mutations in rodent TRPML3, that kill hairy cells
in the inner ear, having adverse effects on hearing (deafness) and
causing impaired balance, as well as resulting in a disruption in
normal pigmentation apparently attributable to the loss of
melanocytes as a result of this same TRPML3 mutation).
[0003] Also, results obtained by the inventors disclosed herein,
and previously unknown, suggest that this gene, because of its
probable role in sodium transport, excretion and absorption, its
role in sodium sensing, and based upon the tissues where it is
known to be specifically expressed including the adrenal gland and
the pituitary gland, indicate that it modulates or participates
with aldosterone and/or vasopressin in the regulation of sodium
transport, metabolism, excretion and other sodium and possibly
other ion related functions involving aldosterone and/or
vasopressin as these hormones are play an important role in sodium
transport, metabolism, and excretion in different mammals including
humans and rodents.
FIELD OF THE INVENTION
[0004] This invention relates to the discovery that a specific ion
channel polypeptide (TRPML3 or MCOLN3) which is member of the TRPML
subfamily of the transient receptor potential cation channel
superfamily is involved in taste (salty) perception, e.g., sodium
taste sensing.
[0005] More specifically, this invention relates to the discovery
that a specific ion channel polypeptide (TRPML3 or MCOLN3) is
involved in taste (salty) perception in primates (human and
non-human primates), rodents, and other mammals, and likely other
vertebrates (avians, reptiles, amphibians), given the importance of
maintaining sodium and potassium ion levels within different
concentration thresholds, and given the important effect of these
ions on physiological processes important to the well being of
different organisms.
[0006] In addition, the invention relates to the discovery that
TRPML3 or MCOLN3 polypeptides or functional variants thereof when
expressed separately or in combination with other taste specific
polypeptides (e.g., other accessory molecules such as GPCRs or ion
channels including e.g., TRPML1, TRPML2, NKAIN3 or NALCN) functions
as a taste receptor that responds to salty taste stimuli and
potentially other taste eliciting molecules.
[0007] In addition the invention relates to the discovery that this
gene, because of its role in sodium sensing, and the tissues where
it is known to be specifically expressed, which include the adrenal
and the pituitary gland, encodes a polypeptide that alone or in
association with other accessory molecules plays a role in
modulating the levels and release of aldosterone and vasopressin
and thereby sodium related physiological activities regulated by
aldosterone and/or vasopressin.
[0008] In addition the invention relates to the discovery that
TRPML3 modulators, because TRPML3 is involved in sodium sensing,
and further based on its expression in the parathyroid organ can be
used to treat diseases involving the parathyroid including but not
limited to calcium homeostasis, hyperparathyroidism,
hypoparathyroidism, hypercalcemia, osteitis cystica,
pseudoparathyroidism, Jansen's metaphyseal chondroplasia,
Blomstrand's chondroplasia, and osteoporosis of different causes
including e.g., age related, menopausal, or drug, chemotherapy or
radiotherapy induced.
[0009] The invention also relates to the use of an animal model
(Varitint waddler mouse) containing a mutated form of TRPML3 or
MCOLN3 gene, where TRPML3 salty taste cells are specifically
ablated from taste buds and where salty taste is greatly diminished
in order to study the effect of TRPML3 on salty taste and other
effects of this ion channel on sodium transport, metabolism, and
excretion, as the gene likely has the same effects in other animals
including humans.
[0010] The invention also relates to the use of a mutant TRPML3
gene (A419P TRPML3) to specifically ablate cell types including
taste cells or melanocytes and create mouse model systems lacking
these different cell populations.
[0011] The invention also relates to the use of a mutant TRPML3 ion
channel polypeptide (A419P TRPML3) as a toxin to kill specific cell
types such as salty taste cells or melanocytes.
[0012] The invention also relates to applications of this gene and
the corresponding polypeptide or variants thereof including allelic
variants, chimeras, fragments and engineered mutants (e.g., mutants
designed to modulate (increase or decrease) activity or to maintain
ion channel in open or closed position) in assays for identifying
TRPML3 modulatory compounds (TRPML3 agonists, antagonists,
enhancers, blockers). These compounds potentially may be used as
taste modulators and therapeutics that modulate or treat
physiological functions and conditions involving aberrant
vasopressin release, aldosterone production, melanocyte function or
sodium transport, absorption and/or excretion. For example these
compounds may be used as salty taste blockers, enhancers, or
inhibitors, or for treating hypertension, urinary function,
cardiovascular diseases, for treating melanocyte related conditions
such as pigmentation disorders, melanomas, and mucous related
conditions such as mucolipidosis type IV.
[0013] Based on the foregoing, the present invention specifically
relates to a mammalian cell-based high-throughput screening assays
(HTS assays) for the identification of TRPML3 modulators. In an
exemplary embodiment, the invention teaches the use of cells
expressing an active variant of TRPML3 (A419P-TRPML3) in cell-based
assays for the identification of enhancers or blockers of TRPML3
function. Compounds that modulate TRPML3 function in a cell-based
assay are anticipated to affect salty taste in humans and other
mammals. The assays described in the present invention can be run
in standard 96 or 384 well culture plates in high-throughput
mode.
[0014] In an more specific embodiment this invention identifies and
provides functional (electrophysiological), in vivo and
immunohistochemistry data which provide evidence that TRPML3
(MCOLN3) encodes a polypeptide that functions as a primate (e.g.,
human) salty taste receptor
[0015] In a related embodiment the present invention provides the
use of TRPML3 polypeptides and nucleic acid sequences and probes
specific thereto as markers which can be used to enrich, identify
or isolate salt receptor and other TRPML3 expressing cells.
[0016] In a related embodiment the present invention provides the
use of TRPML3 polypeptides and nucleic acid sequences and probes
specific thereto as markers which can be used to identify mutations
in TRPML3 that may correlate to specific diseases and conditions
relating to aberrant TRPML3 function or expression such as diseases
involving abnormal sodium sensing, transport, excretion and
absorption, melanocyte function (cancer, pigmentation disorders, et
al.) and diseases involving aberrant aldosterone or vasopressin
production or release such as cardiovascular and urinary
diseases.
[0017] In another specific aspect this invention provides in vitro
and in vivo assays which use TRPML3 (MCOLN3) and TRPML3 expressing
cells or TRPML3 transgenic animal models to identify agonist,
antagonist or enhancer compounds which elicit or modulate (block or
enhance) salty taste in primates including humans. These assays use
cells or animals which express TRPML3 (wild type or a variant
thereof) or use cells which express the TRPML3 ion channel or a
variant (e.g., functional variant having enhanced activity or
genetically engineered to be fixed in the "open" or "closed"
orientation) in association with other accessory polypeptides such
as other taste specific polypeptides such as NALCN or NKAIN3, GPCRs
or related ion channels such as TRPML1 and/or TRPML2.
[0018] In another aspect this invention provides transgenic
animals, preferably rodents, and the use thereof to confirm the
role of TRPML3 in salty taste in mammals and in other physiological
functions involving sodium and other ions such as potassium,
calcium, lithium and on ion (sodium) metabolism, blood pressure,
fluid retention and excretion, urinary function and cardiac
function.
[0019] In another aspect this invention provides in vitro and in
vivo assays which use TRPML3 and TRPML3 expressing cells or
transgenic animals in assays, e.g., neurophysiological behavioral
or electrophysiological assays, in order to identify therapeutic
compounds which alleviate diseases and conditions involving or
caused by deficiencies in the expression of this ion channel
polypeptide and/or its effects on specific cells including
ablation. These conditions include by way of example conditions
involving TRPML3 hyperexpression, hypoexpression, and mutations in
the TRPML3 polypeptide that affect its ability to function as a
taste specific sodium channel in a mammal including e.g., human and
non-human primates. Other conditions include by way of example
Addison's disease and diseases involving aberrant aldosterone or
vasopressin production or release such as hypertension,
hypotension, fluid retention, and impaired urinary or cardiac
function such as arrhythmia, heart attack and stroke.
[0020] In another embodiment the invention relates to assays that
identify compounds that modulate the function of the use of TRPML3
alone or expressed in association with another taste specific gene
such as NALCN or NKAIN3 or TRPML1 or TRPML2 and the use of the
identified compounds to modulate salty taste perception in humans
and other mammals or vertebrates. In another embodiment the
invention relates to assays that identify compounds that modulate
the function of the use of TRPML3 alone or expressed in association
with another taste specific gene such as NALCN or NKAIN3 or TRPML1
or TRPML2 and the use of the identified compounds to modulate salty
taste perception in humans and other mammals or vertebrates.
[0021] The present invention further provides specific methods of
isolating, purifying and marking salty and other TRPML3 expressing
cell types and taste cell lineages including as well as taste stem
cells and other immature and mature taste cell lineages including
cells that differentiate into taste bud cells, taste cell neurons,
taste immune cells et al. based on the expression or absence of
expression of TRPML3 identified using the methods provided herein.
These isolation and purification methods include both positive and
negative cell separation methods. For example desired taste cell
lineages or types may be isolated by positive cell selection
methods e.g., by the use of fluorescence activated cell sorting
(FACS), magnetic bead cell selection e.g., by visual identification
of desired cells such as individual transfected cells by
electrophysiology using antibody coated beads. Alternatively,
desired taste cell lineages or types may be recovered or purified
by negative cell purification and isolation methods wherein the
desired cell types are enriched or purified from a mixed cell
population by the removal of one or several undesired cell lineages
e.g., by contacting a mixed cell suspension containing the desired
taste cells and undesired cells e.g., derived from the tongue, oral
cavity or gastrointestinal tract and associated organs with
cytotoxic antibodies specific to a target gene or genes expressed
on the undesired taste cell type(s) which are to be removed.
[0022] Also, the invention relates to the use of the Varitint
waddler mice to detect the effect of TRPML3 function on
melanocytes, pituitary, adrenal, taste, urinary or taste cells.
[0023] Also, the invention relates to the use of the Varitint
waddler mice to detect the effect of TRPML3 function on
melanocytes, pituitary, adrenal, taste, urinary or taste cells.
[0024] In addition the invention relates to the discovery that
TRPML3 modulators, because TRPML3 is involved in sodium sensing,
and further based on its expression in the parathyroid organ can be
used to treat diseases involving the parathyroid including but not
limited to calcium homeostasis, hyperthyroidism, hypothyroidism,
hypercalcemia, osteitis cystica, pseudothyroidism, Jansen's
metaphyseal chondroplasia, Blomstrand's chondroplasia, and
osteoporosis of different causes including e.g., age related,
menopausal, or drug, chemotherapy or radiotherapy induced.
[0025] Also, the invention relates to the use of the Varitint
waddler mice in assays to detect genes specifically expressed in
salty taste cells and not in the Varitint waddler mice (as salty
taste cells are ablated therein) which genes may modulate TRPML3
function, or function as a salty taste receptor or modulate
transmission of salty taste signaling from TRPML3 to the nerve
fibers and/or control the development differentiation or apoptosis
of salty taste cells. These gene detection assays may comprise the
use of gene chips or microarray technology to compare the genes
expressed in salty taste cells versus genes expressed in Varitint
waddler mice.
[0026] The present invention further provides specific methods of
isolating, purifying and marking salty and other TRPML3 expressing
cell types and taste cell lineages including as well as taste stem
cells and other immature and mature taste cell lineages including
cells that differentiate into taste bud cells, taste cell neurons,
taste immune cells et al. based on the expression or absence of
expression of TRPML3 identified using the methods provided herein.
These isolation and purification methods include both positive and
negative cell separation methods. For example desired taste cell
lineages or types may be isolated by positive cell selection
methods e.g., by the use of fluorescence activated cell sorting
(FACS), magnetic bead cell selection e.g., by visual identification
of desired cells such as individual transfected cells by
electrophysiology using antibody coated beads. Alternatively,
desired taste cell lineages or types may be recovered or purified
by negative cell purification and isolation methods wherein the
desired cell types are enriched or purified from a mixed cell
population by the removal of one or several undesired cell lineages
e.g., by contacting a mixed cell suspension containing the desired
taste cells and undesired cells e.g., derived from the tongue, oral
cavity or gastrointestinal tract and associated organs with
cytotoxic antibodies specific to a target gene or genes expressed
on the undesired taste cell type(s) which are to be removed. Also,
the invention relates to the use of the Varitint waddler mice to
detect the effect of TRPML3 function on melanocytes, pituitary,
adrenal, taste, urinary or taste cells.
[0027] Also, the invention relates to the use of the Varitint
waddler mice to detect the effect of TRPML3 function on
melanocytes, pituitary, adrenal, taste, urinary or taste cells.
[0028] Also, the invention relates to the use of the Varitint
waddler mice in assays to detect genes specifically expressed in
salty taste cells and not in the Varitint waddler mice (as salty
taste cells are ablated therein) which genes may modulate TRPML3
function, or function as a salty taste receptor or modulate
transmission of salty taste signaling from TRPML3 to the nerve
fibers o and/or control the development differentiation or
apoptosis of salty taste cells. These gene detection assays may
comprise the use of gene chips or microarray technology to compare
the genes expressed in salty taste cells versus genes expressed in
Varitint waddler mice.
[0029] Also the invention relates to the use of markers e.g.,
antibodies or oligonucleotides, that are specific to TRPML3 and/or
related accessory polypeptides in mapping regions of the tongue and
oral cavity which are involved in specific taste (salty) and
non-taste specific functions, mapping of cell comprised on specific
(salty) taste sensing cells in the gastrointestinal tract and
associated organs such as the intestinal epithelium or urinary
tract that express specific taste specific genes and which
therefore are involved in one or more of the taste cell specific
functions disclosed herein, and/or the use of the subject genes and
markers specific thereto in taste cell differentiation studies,
e.g. for identifying compounds that induce the differentiation or
dedifferentiation of taste cells e.g., adult or embryonic stem
cells and other pluripotent or immature cell types into desired
taste cell lineages and taste cell types.
BACKGROUND OF THE INVENTION
[0030] Various ion channels have been studied in order to elucidate
their potential involvement in salty taste and regulation of sodium
transport, metabolism and excretion. In particular, epithelial
sodium channels (ENaC) which are members of the ENaC/degenerin
family of ion channels that includes acid-sensing ion channels
(ASIC) in mammals, mechanosensitive degenerin channels in worms,
and FMRF-amide peptide-gated channels in mollusks (Kellenger, S.
and Schild, L. (2002) Physiol. Rev. 82:735-767) have been
extensively studied. ENaC mediates amiloride-sensitive apical
membrane Na.sup.+ transport across high resistance epithelia in
numerous tissues including kidney, colon, and lung.
[0031] ENaC is known to be a heterotrimeric channel comprised of
alpha, beta, and gamma subunits or delta, beta, and gamma subunits.
This heterotrimeric channel has been hypothesized to be involved in
human salty taste perception. Previously, assays have been
developed by the present assignee using ENaC sequences to identify
compounds that modulate the delta beta gamma and alpha beta gamma
human ENaC to examine if these compounds will potentially modulate
human salty taste perception. Also, these compounds potentially may
be used to treat human pathologies involving aberrant ENaC
function.
[0032] Unlike other mammals, amiloride only slightly reduces the
intensity of sodium chloride taste, i.e., by about 15-20% when used
at concentrations that specifically modulate ENaC function
(Halpern, B. P. (1998) Neuroscience and Behavioral Reviews. 23:
5-47). Experiments conducted by the inventors have shown that
amiloride, or the more potent amiloride derivative phenamil did not
elicit a significant effect on perceived human salt intensity when
tested at levels 300-fold (for amiloride) and 3000-fold (for
benzamil) above IC50 values for alpha beta gamma ENaC (equivalent
to 10-fold for amiloride and 100-fold for benzamil over IC50 values
for delta beta gamma ENaC). Based thereon, it was theorized that
other (ENaC genes) were involved in human salty taste
perception.
[0033] In addition, it has been recently reported that taste
receptors may be expressed in non-oral tissues, e.g., in the
digestive system and potentially other organs such as the kidney,
suggesting that they have non-taste related activities, such as in
food sensing and regulation of digestion et al. Particularly it has
been reported that sweet, umami and bitter taste receptors are
expressed in cells other than in the oral cavity such as
gastrointestinal cells. (See, e.g., Sternini et al., Amer J
Physiol. Gastrointestinal and Liver Physiology, 292:G457-G461,
2007; Mace, O. J. et al, J. Physiology.
10.1113/jphysiol.2007.130906. Published online May 10, 2007). Also,
it has been reported by various groups (Margolskee et al., Bezencon
et al., Rozengurt et al, and Sternini et al. (2007) (Id)) that
bitter and umami taste receptors and other taste signaling
molecules such as TRPM5 and gustducin are expressed in specialized
cells in the gastrointestinal tract. (See e.g., Margolskee et al.,
Genes Brain Behavior 2007 (epub March 21); Rozengurt et al., Amer.
J. Physiol. Gastroent. Liver Physiol. 291(2):G171-7 (2006);
Bezencon et al., Chem Senses 32(1):41-47 (2007)). Margolskee et al.
(Id) further reports that the loss of T1R3 or gustducin in rodents
resulted in changes in insulin metabolism and the release of
satiety peptides such as GLP-1 (glucagon-like peptide 1).
[0034] Based on these observations with other taste receptors, it
is likely that salty receptors are expressed in tissues that play
important roles in controlling sodium ion homeostasis such as the
adrenal gland and pituitary gland. Because taste receptors are
expressed on non-taste cells such as digestive organs and likely
organs in the urinary system they are likely involved in functions
not directly related to taste such as digestive functions such as
gastric motility, absorption, food detection, metabolism, and
immune regulation of the oral or digestive tract and may also
affect functions relating to sodium absorption, excretion and
transport such as blood pressure and fluid retention. Therefore,
the identification of taste cell specific genes and identifying
what specific cells these genes are specifically expressed should
facilitate a better understanding of other non-taste functions of
these taste receptors and also facilitate the use of these genes,
gene products and cells which express same in assays for
identifying novel therapeutics, e.g., for treating digestive
diseases such as autoimmune, inflammatory and cancers, metabolism,
diabetes, eating disorders, obesity, taste cell turnover,
hypertension, fluid retention, and immune regulation of the
digestive system. In the specific case of sodium (salty) taste
receptors, elucidating the specific identity of the gene or genes
which are significant for salty taste sensing should facilitate an
understanding of the role of these genes on other sodium related
functions and polypeptides such as vasopressin or aldosterone which
are involved in sodium transport, metabolism and excretion,
critical to urinary and cardiovascular function.
[0035] As mentioned above, this invention relates specifically to
the discovery that TRPML3 is an ion channel polypeptide that is
involved in sodium (salty) taste sensing in mammals and potentially
other vertebrates given the importance of sodium and other ions
(such as potassium, calcium, lithium) to many physiological
functions which would indicate that this gene may be conserved in
different vertebrates. Prior to the specific discovery of the
inventors herein, i.e. that TRPML3 (MCOLN3) encodes an ion channel
that is involved in salty taste perception in primates and other
mammals, (and further likely plays a role in related physiological
functions involving sodium transport and excretion), this gene had
been reported previously to be responsible for the phenotype of a
mouse mutant called varitint-waddler that exhibits early-onset
hearing loss, vestibular defects, pigment abnormalities and
perinatal lethality (DiPalma et al., Mutations in Mcoln3 associated
with deafness and pigmentation defects in varitint-waddler (Va)
mice. Proc. Natl. Acad. Sci. USA 99: 14994-14999; 2002). It was
further reported that MCOLN3 or TRPML3 is expressed in the hair
cells and plasma membrane of stereocilia (in the ears).
Particularly, a mutation in this polypeptide that resulted in an
ala 419 to pro substitution in the fifth transmembrane domain has
been reported to result is a hyperactive MCOLN3 that results in the
death of cells expressing this molecule, i.e., the hair cells of
the ear (hence the deafness of the Va mouse) (Grim C et al., Proc
Natl. Acad. Sci. USA 104:19583-8; 2007).
[0036] While in mice, the A419P-TRPML3 mutation results in a severe
form of the varitint-waddler phenotype. (Kim et al., J. Biol.
Chem., 2007 Dec. 14; 282(50):36138-42. Epub 2007 Oct. 25; Nagata et
al., Proc. Natl. Acad. Sci, USA, 2008 Jan. 8; 105(1):353-8. Epub
2007 Dec. 27; Grimm et al., Proc. Natl. Acad. Sci, USA, 2007 Dec.
4, 104(49):19583-8. Epub 2007 Nov. 28), characterized by
pigmentation defects, hearing loss and embryonic lethality, another
TRPML3 mutation (TRPML3(I362T/A419P) results in a milder form of
the varitint-waddler phenotype.
[0037] How the mutations cause each phenotype are not known. It has
been reported that the first channel properties of TRPML3 are as a
strongly inward rectifying cation channel with a novel regulation
by extracytosolic Na+. (Kim et al. 2007 (Id.)) They further report
that preincubating the extracytosolic face of TRPML3 in Na+-free
medium is required for channel activation, but then the channel
slowly inactivates. Therefore, the A419P mutation locks the channel
in an open unregulated state. The Kim et al. researchers further
observed similar gain of function with the A419G mutation, which,
like A419P, is expected to destabilize the alpha-helical fifth
transmembrane domain of TRPML3. By contrast, Kim et al., observed
that the I362T mutation results in an inactive channel, but the
channel properties of TRPML3(I362T/A419P) were similar to those of
TRPML3(A419P). However, they reported that the surface expression
and current density of TRPML3(I362T/A419P) are lower than those of
TRPML3(A419P) and that the A419P mutation reportedly affects
channel glycosylation and causes massive cell death. Their findings
reportedly further confirmed that the varitint-waddler phenotype is
due to a gain of function of TRPML3(A419P) that is reduced by the
TRPML3(I362T/A419P) mutant, resulting in a milder phenotype.
[0038] In addition, it had been reported for a related member of
the TRPML3 gene family, TRPML1 (mucolipin 1/MCOLN1), that some
mutations result in mucolipidosis type IV, a severe inherited
neurodegenerative disease. Xu et al., 2007, Nov. 13 Proc Acad Sci
USA, 104(46):18321-6 Epub 2007 Nov. 7). This disease is a specific
form of mucolipidosis, which is an autosomal recessive lysosomal
storage disorder characterized by severe neurodegeneration,
achlorhydria, and visual impairments such as corneal opacity and
strabismus. The disease arises due to mutations in a group 2
transient receptor potential (TRP)-related cation channel, TRPML1.
(Venkatanulum et al., J. Biol. Chem. 2006 Jun. 23 281(25):17517-27
epub 2006 Apr. 10).
[0039] It has also been reported that the members of the TRPML3
gene family associate with one another. For example the same
reference Venkatanulum et al. (id.) suggests the propensity of
these (TRPML1, 2 and 3) proteins to multimerize, and teaches that
the subcellular distribution and mechanisms that regulate their
trafficking are limited. They also allege that TRPMLs interact to
form homo- and heteromultimers. Moreover, Venkatanulum et al. also
purport that the presence of either TRPML1 or TRPML2 specifically
influences the spatial distribution of TRPML3. They allege that
while TRPML1 and TRPML2 homomultimers are lysosomal proteins, that
TRPML3 homomultimers are in the endoplasmic reticulum. In addition,
they allege that TRPML3 localizes to lysosomes when coexpressed
with either TRPML1 or TRPML2 and is comparably mislocalized when
lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely,
they state that TRPML3 does not cause retention of TRPML1 or TRPML2
in the endoplasmic reticulum. Also, Venkatanulum et al. suggest
that there is a hierarchy controlling the subcellular distributions
of the TRPMLs such that TRPML1 and TRPML2 which dictates the
localization of TRPML3 and not vice versa.
[0040] Also it had been reported in the literature and in public
gene databases that MCOLN3 or TRPML3 is strongly expressed in the
adrenal glands which glands are known to play an important role in
the regulation of sodium metabolism in the body. Further, it had
been reported that a human autoimmune disease (Addison's) is
characterized by the destruction of the adrenal glands and that
this disease has as one of its telltale symptoms strong salt
cravings.
[0041] Still further, it had been reported that TRPML3 is strongly
expressed in the pituitary glands, and is expressed in melanocytes.
As noted above, the varitint mutation, as well as resulting in the
death of hearing cells, results in the death of melanocytes.
[0042] However, to the best of the inventors' knowledge no one had
previously suggested that TRPML3 or the related genes TRPML1 or
TRPML2 as being involved in salty taste or to encode a taste
receptor polypeptide that senses and responds to salty taste
stimuli in different mammals or other vertebrates.
BRIEF DESCRIPTION AND OBJECTS OF THE INVENTION
[0043] Therefore, it is an object of the invention to provide the
discovery and supporting data which establishes for the first time
that TRPML3 plays an active role in taste, specifically salty taste
in different mammals and potentially other vertebrates and that
based thereon this ion channel, alone or in association with other
ion channel genes such as TRPML1, TRPML2, or NALCN, NKAIN3 or other
accessory proteins may further play a significant role in sodium
related cellular and physiological activities such as sodium
absorption, transport, and excretion and related ancillary effects
and activities such as urine output, blood pressure regulation, and
the like.
[0044] It is also an object of the invention to provide transformed
or transfected cells or transgenic animals that express TRPML3 or
variants thereof and optionally other ion channels or accessory
proteins such as taste specific GPCRs suitable for use in assays in
identifying TRPML3 modulators or for study of the effect of TRPML3
on salty taste and other physiologic processes involving sodium
transport, metabolism, and excretion.
[0045] Also, it is an object of the invention to provide assay
systems that comprise test cells, preferably recombinant mammalian
cells or amphibian oocytes, or endogenous TRPML3 expressing salty
taste cells or other TRPML3 expressing cells (e.g., pituitary or
adrenal) that express a functional TRPML3 or a variant, fragment or
functional equivalent as well as mammalian cell-based and amphibian
oocyte cell-based assays, preferably high-throughput, for putative
modulators of TRPML3.
[0046] More specifically, it is an object of the invention to
provide human cell lines, e.g., HEK293T cells, CHO cells and
amphibian oocytes, that express a functional TRPML3 or a variant,
fragment or functional equivalent that can be used in cell-based
assays to screen for TRPML3 modulators.
[0047] Also, it is an object of the invention to provide mammalian
cells and amphibian oocytes that express functional TRPML3 or a
variant, fragment or functional equivalent for use in functionally
characterizing TRPML3 activity, and to identify compounds that
either enhance or block salty taste perception (herein referred to
as salty taste modulators). These compounds can be used as
ingredients in foods, medicinals and beverages to enhance,
modulate, inhibit or block salty taste.
[0048] As disclosed in the provisional applications and n earlier
utility patent application to which this application claims benefit
of priority, the inventors identified this gene initially as
encoding a taste specific gene using a novel rationale for
identifying primate taste specific genes (disclosed in detail in
the provisional applications incorporated by reference herein).
These applications showed that this gene which encodes a
multitransmembrane protein is expressed in the top of the taste
buds, in the taste sensory cells, that conducts sodium. It was
conjectured based thereon that TRPML3 may be involved in salty
taste perception and other sodium related functions.
[0049] Data disclosed herein including functional
(electrophysiological) data both in vitro and in vivo (using
varitint-waddler mouse which expresses a mutant form of TRPML3 gene
that causes deafness, pigment loss and impaired balance),
immunohistochemical data, and other information provided infra
substantiate the inventors' original supposition and provide
convincing experimental validation that this gene encodes a salt
receptor that allows sensory taste cells in the tongue's taste buds
to detect sodium chloride (salt).
[0050] In addition, based on the fact that this gene is also
expressed in the adrenal and pituitary glands, it is anticipated to
participate or regulate in association with other polypeptides such
as vasopressin and aldosterone the general regulation of sodium
transport, metabolism, and excretion in different cells, tissues
and organs in the body.
[0051] The evidence contained herein which in combination provide
convincing evidence that the TRPML3 gene encodes an ion channel
that functions as a salty taste receptor in different animals
include the following:
[0052] (1) MCOLN3 or TRPML3 is specifically expressed at least in
primate and human taste and not lingual epithelial cells and is
specifically expressed in the top of the taste buds, in a subset of
taste sensory cells that do not express TRPM5 (that is, they are
not sweet, bitter or umami), do not express PKD2L1 (that is, they
are not sour) and are found towards the taste pore. Therefore
MCOLN3 positive cells comprise a separate subset of taste cells
distinct from known taste cells involved in detecting other
(non-salt) taste modalities.
[0053] (2) MCOLN3 or TRPML3 is also expressed in sensory cells of
other organs, like the ear. It is therefore a `professional`
sensory gene.
[0054] (3) MCOLN3 or TRPML3 is strongly expressed in the adrenal
glands. These glands play a very important role in the regulation
of sodium metabolism in the body. MCOLN3 is therefore likely (based
on this and other data obtained by the inventors) to be a key
molecule in the regulation of sodium metabolism and may regulate or
participate in the production of aldosterone by the adrenal
glands.
[0055] (4) A human autoimmune disease (Addison's) is characterized
by the destruction of the adrenal glands. One of the telltale
symptoms of this disease is salt craving. The latter is likely the
result of the presence of autoantibodies against MCOLN3, or a
mutation in this gene that disrupt the function of MCOLN3 or TRPML3
in taste buds.
[0056] (5) MCOLN3 or TRPML3 is also highly expressed in the
pituitary glands which are involved in vasopressin release, which
in turn affects urine production and kidney function. Based
thereon, and other data obtained by the inventors relating to
TRPML3, this polypeptide likely regulates vasopressin release and
thereby sodium excretion in the urine.
[0057] (6) MCOLN3 or TRPML3 conducts sodium in electrophysiology
studies and exhibits the right biochemical characteristics
predicted for a primate salty taste receptor (i.e., the detection
of K+, Li+ and amiloride insensitivity).
[0058] (7) Neurophysiological experiments (nerve recordings) using
sodium in the varitint mouse (having TRPML3 mutation) indicate that
the Varitint mouse is impaired in its response to sodium (does not
exhibit a robust salty taste response). These mice are ablated of
the TRPML3 expressing taste cells (salty taste cells) in the taste
bud confirming that these specific cells are a prerequisite for
detection of salty taste.
[0059] (8) Cell based assays using mammalian cells and amphibian
oocytes which express mutated TRPML3 polypeptides (mutation results
in the ion channel being fixed in the "open" orientation) have
identified TRPML3 enhancers and blockers which should enhance or
block salty taste in taste tests.
[0060] Therefore, based on the foregoing it is an object of the
invention to establish the identity of MCOLN3 or TRPML3 as a human
salty taste receptor and based thereon to design screening assays
using cells or animals transfected with this gene or a variant
(e.g., functional chimera, mutant possessing enhanced activity,
fixed in open orientation or other desired change in TRPML3 protein
facilitating its use in assays) for the purpose of identifying
agonists, antagonists or enhancers (modulators) of the function of
this molecule which will modulate salty taste and other TRPML3
functions.
[0061] Also, it is an object of the invention to provide an
isolated taste, adrenal, pituitary or urinary organ cell or
enriched cell sample comprising a taste, adrenal, pituitary or
urinary organ cell that expresses TRPML3 that is involved in salty
taste perception, sodium metabolism, aldosterone production, and/or
vasopressin release wherein said isolated taste, adrenal,
pituitary, or urinary organ cell or enriched taste cell sample
expresses TRPML3 gene or a variant thereof that encodes a sodium
channel that modulates at least one of salty taste, sodium
transport, metabolism, or excretion and/or aldosterone or
vasopressin release or production. Preferably the cell will be of
human, non-human primate or rodent origin.
[0062] Also, it is a specific object of the invention to identify
TRPML3 modulators which will be useful in treating diseases
involving aldosterone release or production. Diseases and
conditions treatable using TRPML3 modulators include diseases
treatable by compounds which agonize or antagonize aldosterone and
thereby sodium transport and excretion and include by way of
example edema, blood pressure (hyper or hypotension), liver
cirrhosis, primary hyperaldosteronemia, renal dysfunction, diabetes
(Type I or II) and the pathological symptoms associated therewith
including circulatory problems, edema, ocular disorders relating to
poor circulation, hypercortisolaemia, atherosclerosis or obesity,
e.g., abdominal obesity, as well as liver disease, sexual
dysfunction (male or female), cerebrovascular disease, vascular
disease, retinopathy, neuropathy, insulinopathy, endothelial
dysfunction, baroreceptor dysfunction, migraine headaches, hot
flashes, and premenstrual tension and other cardiovascular
conditions such as atherosclerosis, heart failure, congestive heart
failure, vascular disease, stroke, myocardial infarction,
endothelial dysfunction, ventricular hypertrophy, renal
dysfunction, target-organ damage, thrombosis, cardiac arrhythmia,
plaque rupture and aneurysm.
[0063] Also, it is a specific object of the invention to identify
TRPML3 modulators which will be useful in treating diseases
involving vasopressin release or production. Diseases and
conditions treatable using TRPML3 modulators include diseases or
conditions treatable by compounds that agonize or antagonize
vasopressin and similarly include by way of example diabetes,
obesity, kidney diseases such as cystic kidney disease, acquired
renal cystic disease, ocular circulation related disorders such as
myopia; nausea, emesis, sexual dysfunction (male or female), edema,
hypertension, congestive heart failure (ranging from class II of
the New York Heart Association to florid pulmonary edema), periodic
idiopathic edema, nephrotic syndrome, ascites due to cirrhosis or
other causes, cerebral edema of various causes, as well as
dilutional hyponatremia and metabolic alterations collectively
known as the syndrome of inappropriate ADH secretion and other
diseases or conditions wherein vasodilation and/or antioxytocic
activity is therapeutically desirable.
[0064] Also more specifically, it is an object of the invention to
provide an isolated taste receptor that modulates salty taste
perception comprising a TRPML3 polypeptide or variant thereof such
as one engineered to possess enhanced ion channel activity or to
remain fixed in the open orientation or a chimera or fragment of
TRPML3 that modulates salty taste in mammals including humans. This
taste receptor may be monomeric or polymeric (heteropolymeric or
homopolymeric) and may comprise other taste specific polypeptides,
e.g., other ion channel polypeptides such as TRPML2, TRPML1, NKAIN3
or NALCN.
[0065] Also more specifically, it is an object of the invention to
provide a transgenic non-human animal which has been genetically
engineered to knock out the expression of endogenous TRPML3 and/or
to further genetically engineer the knocking in of an ortholog or
variant thereof, e.g. one engineered to enhance ion channel
activity or to fix the channel in the "open" position. These
animals, including those expressing human or other primate TRPML3
genes or variants may be used to identify compounds that modulate
(enhance or block) salty taste in humans and other mammals.
[0066] More specifically, it is an object of the invention to
provide a transgenic non-human animal which has been genetically
engineered to express a heterologous TRPML3 polypeptide, e.g., a
human TPML3 or variant.
[0067] Also more specifically, it is an object of the invention to
provide assay methods of using a transgenic animal that expresses
TRPML3 or a mutant form such as the mutation causing the
Varitint-waddler phenotype in screens to identify antagonists,
agonists or enhancers of TRPML3 and to study the effects of TRPML3
on different physiological activities including salty taste and
sodium transport, metabolism and excretion.
[0068] Also more specifically, it is an object of the invention to
provide methods of using a transgenic animal according wherein the
TRPML3 gene has been "knocked out" in order to elucidate the effect
of TRPML3 on taste, and on cardiac or urinary function and in
particular on aldosterone production, sodium metabolism, salty
taste perception or vasopressin release. It is anticipated that
these animals may comprise conditions relating to sodium transport
and metabolism such as hypertension, hypotension, fluid retention,
heart attack and stroke. Therefore, the invention further includes
the use of these animals as disease models and for evaluation of
potential therapeutics for treating or preventing such
conditions.
[0069] Also, more specifically it is an object of the invention to
use mutant forms of TRPML3 polypeptide, including the mutated form
that gives rise to the Varitint-Waddler phenotype, in order to kill
or ablate specific cells including salty taste cells, melanocytes,
pituitary or adrenal cells.
[0070] Also, more specifically it is an object of the invention to
use mutant forms of the gene encoding the TRPML3 polypeptide,
including the mutated form that gives rise to the Varitint-Waddler
phenotype in order to create transgenic animals wherein specific
cells are ablated and to use these transgenic animals in order to
test potential therapeutics and as disease models.
[0071] Also, more specifically it is an object of the invention to
use mutant forms of TRPML3 polypeptide, including the mutated form
that gives rise to the Varitint-Waddler phenotype as toxins to kill
specific cells.
[0072] Also it is an object of the invention to provide the use of
molecules that modulate or bind TRPML3, e.g., which agonize or
antagonize or specifically bind to this polypeptide for the
treatment of melanoma, adrenal cancer, pituitary cancer, et al. and
other conditions involving melanocytes such as pigmentation
disorders or pituitary or adrenal related disorders.
[0073] Also more specifically, it is an object of the invention to
provide a recombinant cell which expresses a salty taste receptor
comprising TRPML3 or a variant thereof that encodes a functional
sodium ion channel polypeptide.
[0074] Also more specifically, it is an object of the invention to
provide an assay for identifying compounds that agonize, antagonize
or enhance an activity of TRPML3 comprising contacting a
recombinant or endogenous taste or other cell that expresses TRPML3
with a putative TRPML3 enhancer, agonist or antagonist and
determining the effect thereof on TRPML3 activity. Preferably these
assays will be electrophysiological assays e.g., patch clamp or two
electrode voltage clamping assays, which may be automated and
typically will use mammalian or amphibian cells.
[0075] Also more specifically, it is an object of the invention to
provide methods for identifying TRPML3 modulators by an ion flux
assay.
[0076] Also more specifically, it is an object of the invention to
provide methods for identifying TRPML3 modulators (enhancers,
blockers) by an automated electrophysiological (patch clamp) assay,
i.e., IonWorks assay system
[0077] Also more specifically, it is an object of the invention to
provide methods for identifying TRPML3 modulators (enhancers,
blockers) by electrophysiological assays using frog oocytes.
[0078] Also more specifically, it is an object of the invention to
provide methods for identifying TRPML3 modulators (enhancers,
blockers) by electrophysiological assays using mammalian cells.
[0079] Also more specifically, it is an object of the invention to
provide such TRPML3 assays wherein the identified agonist,
antagonist, or enhancer compounds are evaluated in a taste
test.
[0080] Also more specifically, it is an object of the invention to
provide such TRPML3 assays wherein the effect of the identified
agonist, antagonist, or enhancer compounds on aldosterone
production is tested in an animal.
[0081] Also more specifically, it is an object of the invention to
provide such TRPML3 assays wherein the effect of the identified
agonist, antagonist, or enhancer compounds on vasopressin release
is tested in an animal.
[0082] Also more specifically, it is an object of the invention to
provide such TRPML3 assays wherein the effect of the identified
agonist, antagonist, or enhancer compounds on at least one of
cardiac or urinary function and more specifically on blood
pressure, fluid retention, sodium metabolism or urine production,
wherein this is tested in an animal.
[0083] Also more specifically, it is an object of the invention to
provide the use of the identified agonist, antagonist, or enhancer
compounds for treating a disease or condition involving aldosterone
production comprising administering an effective amount of a
compound that modulates TRPML3.
[0084] Also more specifically, it is an object of the invention to
provide the use of the identified agonist, antagonist, or enhancer
compounds for treating a disease or condition involving vasopressin
release comprising administering an effective amount of a compound
that modulates TRPML3.
[0085] Also more specifically, it is an object of the invention to
provide the use of the identified agonist, antagonist, or enhancer
compounds for modulating cardiac function, e.g., blood pressure,
arrhythmia, or stroke or fluid retention in a subject in need
thereof comprising administering an effective amount of a compound
that modulates TRPML3.
[0086] Also, more specifically it is an object of the invention to
provide isolated taste, adrenal, pituitary, melanocyte, or urinary
organ cells or an enriched taste cell sample wherein said isolated
or enriched cell sample comprises cells that express a TRPML3 ion
channel polypeptide.
[0087] Also, more specifically it is an object of the invention to
provide an isolated taste receptor that modulates salty taste
perception comprising a TRPML3 polypeptide or variant thereof that
modulates salty taste in mammals.
[0088] Also, more specifically it is an object of the invention to
provide a transgenic non-human animal which has been genetically
engineered to knock out or to impair the expression of endogenous
TRPML3 with the proviso that said transgenic animal is not a
Varitint mouse.
[0089] Also, more specifically it is an object of the invention to
provide a transgenic non-human animal which has been genetically
engineered to express a heterologous TRPML3 polypeptide with the
proviso that said transgenic animal is not a Varitint mouse.
[0090] Also, more specifically it is an object of the invention to
provide a method of using the transgenic animal in screens to
identify salty taste modulating compounds.
[0091] Also, more specifically it is an object of the invention to
provide a method of using the transgenic animal to identify
antagonists, agonists or enhancers of TRPML3 and wherein the
wherein the identified compounds are further optionally evaluated
in human taste tests.
[0092] Also, more specifically it is an object of the invention to
provide a method of using a transgenic animal in order to elucidate
the effect of TRPML3 on aldosterone production, sodium metabolism,
salty taste perception or vasopressin release.
[0093] Also, more specifically it is an object of the invention to
provide a method of using a transgenic animal (non-human) that
expresses a TRPML3 gene that encodes an ion channel that is toxic
to cells which express the ion channel in order to assess potential
therapeutic regimens for diseases or conditions involving aberrant
aldosterone production, vasopressin release, sodium metabolism
and/or melanocyte loss.
[0094] Also, the invention relates to the use of the Varitint
waddler mice to detect the effect of TRPML3 function on
melanocytes, pituitary, adrenal, taste, urinary or taste cells.
[0095] Also, the invention relates to the use of the Varitint
waddler mice in assays to detect genes specifically expressed in
salty taste cells and not in the Varitint waddler mice (as salty
taste cells are ablated therein) which genes may modulate TRPML3
function, or function as a salty taste receptor or modulate
transmission of salty taste signaling from TRPML3 to the nerve
fibers and/or control the development differentiation or apoptosis
of salty taste cells. These gene detection assays may comprise the
use of gene chips or microarray technology to compare the genes
expressed in salty taste cells versus genes expressed in Varitint
waddler mice.
[0096] Also, the invention provides methods of treating parathyroid
related diseases such as calcium homeostasis, hypercalcemia,
osteitis, hypoparathyroidism, hyperparathyroidism, osteitis
fibrosis cystica, pseudoparahypothyroidism, Jansen's metaphyseal
chondroplasia, Blomstrand's chondroplasia, and osteoporosis of
different causes such as diseases, age, menopause, chemotherapy,
radiation therapy, drugs and the like.
[0097] Also, more specifically it is an object of the invention to
provide a recombinant cell which expresses a salty taste receptor
comprising TRPML3 or a variant thereof, e.g., a yeast, amphibian,
insect, bacterial, reptile, avian, or mammalian cell., preferably a
mammalian cell or frog oocyte, such as a CHO-K1, HEK-293, COS, CHO,
BHK cell which may transiently expresses said TRPML3 polypeptide or
stably express said TRPML3 polypeptide.
[0098] Also, more specifically it is an object of the invention to
provide a method of identifying putative salty taste modulators in
a binding assay comprising providing a TRPML3 polypeptide or cell
which expresses TRPML3 and contacting said polypeptide or cell with
putative TRPML3 modulatory compounds and identifying potential
TRPML3 modulators based on their specific binding to TRPML3
polypeptide.
[0099] Also, more specifically it is an object of the invention to
provide a method of modulating blood pressure or fluid retention in
a subject in need thereof comprising administering an effective
amount of a compound that modulates TRPML3.
[0100] Also, more specifically it is an object of the invention to
provide a method of modulating urine production and/or excretion in
a subject in need thereof comprising administering an effective
amount of a compound that modulates TRPML3.
[0101] Also, more specifically it is an object of the invention to
provide a method of treating Addison's disease or type IV
mocolipidosis in a subject in need thereof comprising administering
an effective amount of a compound that modulates TRPML3.
[0102] Also, more specifically it is an object of the invention to
provide specific codon optimized and TRPML3 mutated sequences and
assays using these sequences.
[0103] Also more specifically, it is an object of the invention to
provide the use of such identified agonist, antagonist, or enhancer
compounds for modulating urine production and/or excretion or edema
in a subject in need thereof comprising administering an effective
amount of a compound that modulates TRPML3.
[0104] Also more specifically, it is an object of the invention to
provide such identified agonist, antagonist, or enhancer compounds
which may include polypeptides, antibodies, small molecules,
siRNAs, antisense RNAs, ribozymes et al.
[0105] More specifically, it is an object of the present invention
to provide mammalian and oocyte cell-based assays, preferably high
or medium throughput, for the profiling and screening of a salty
taste receptor (TRPML3) which assays optionally may include the
addition of a compound that modulates TRPML3 function. Such methods
can be used to functionally characterize TRPML3 activity and to
identify the specific motifs or residues required for salt sensing
in different TRPML3 ion channels as well as to identify compounds
that either enhance or block salty taste perception (herein
referred to as salty taste modulators).
[0106] It is also an object of the invention to provide novel
methods for treatment or prevention of conditions relating to
sodium transport and metabolism such as hypertension, hypotension,
fluid retention, heart attack and stroke and conditions mentioned
above by administration of TRPML3 modulators.
[0107] In a specific aspect, the invention provides a method for
identifying a modulator of TRPML3 utilizing a mammalian cell or
oocyte that expresses a functional TRPML3 sodium channel with a
putative TRPML3 modulatory compound, comprising: (i) assaying the
effect of said compound on sodium transport through the TRPML3
channel; and (ii) identifying whether said compound is an TRPML3
modulator based on its enhancing or inhibitory effect on sodium
transport. The invention further comprises (iii) confirming that
the compound identified modulates salty taste in human or mammalian
taste tests. In one embodiment, the TRMPL3 is mammalian TRMPL3. In
yet another embodiment, TRMPL3 is human, non-human primate, rodent
(mouse or rat), cow, pig, horse or sheep TRMPL3.
[0108] In a further embodiment, the in vivo effect of the
identified compound on sodium extraction or urinary function or
cardiovascular or other functions relating to TRPML3 is tested in
humans or mammals. In one embodiment, the TRMPL3 is mammalian
TRMPL3. In yet another embodiment, TRMPL3 is human, non-human
primate, rodent, cow, pig, horse or sheep TRMPL3.
[0109] In one aspect of the present invention, the mammalian cell
is selected from the group consisting of a HEK293, HEK293T,
Swiss3T3, CHO, BHK, NIH3T3, and COS cells. In a second aspect, the
oocyte is a mammalian, amphibian, avian or reptilian oocyte. In a
further aspect, the amphibian oocyte is a Xenopus oocyte. In
another aspect of the invention, the cell expresses an additional
taste gene, preferably an ion channel.
[0110] In a related embodiment of the invention, these assays are
used to identify a human TRPML3 enhancer or inhibitor wherein an
oocyte is contacted with an inhibitor or activator of human TRPML3
prior to contacting with a putative human TRPML3 enhancer. In an
additional embodiment, the assay further comprises a negative
control using an oocyte that has not been microinjected with human
TRPML3 cRNAs. In an additional aspect of the invention, the
putative modulator is applied at a concentration ranging from
around 1 nM to about 1 mM. In another aspect of the invention, the
human TRPML3 enhancer exhibits an enhancement factor of at least
20%. In a further aspect, the human TRPML3 enhancer exhibits an
enhancement factor of at least 50%. In yet a further aspect, the
human TRPML3 enhancer exhibits an enhancement factor of at least
100%.
[0111] Also more generally, it is an object of the invention to
provide a method or rationale for identifying a gene encoding a
polypeptide involved in salty taste perception in a primate (human
or non-human) comprising:
[0112] (i) identifying a set of genes including genes which are
expressed in fungiform and optionally circumvallate, foliate, or
palate taste cells but which are not expressed in lingual cells
and/or genes which are expressed in taste cells at substantially
higher levels than in lingual cells;
[0113] (ii) of the genes identified in (i) optionally identifying a
set of genes which are not expressed in taste cells which express
umami, sweet, bitter, or sour taste receptors or markers of these
cells (T1Rs or T2Rs, TRPM5, and PKD2L1/PKD1L3);
[0114] (iii) optionally identifying a subset of the taste specific
genes contained in the genus of genes identified after step (i) or
step (ii) which are specifically expressed in the top half of taste
buds and not the bottom half of taste buds or which are enriched
(expressed at least 1.2-1.5 fold greater) in the top half than in
the bottom half of taste buds; and
[0115] (iv) functionally expressing one or more genes identified
according to (ii) or (iii) and determining which of said genes
functions as a sodium responsive ion channel or sodium responsive
receptor or transporter and thereby identifying this gene or genes
as a putative gene(s) that modulates salty taste.
[0116] (Preferably, the identified gene which is functionalized is
one which is enriched by at least 1.2-1.5 fold in the top half of
the taste buds relative to the bottom half of the taste buds).
[0117] In these methods step (i) preferably comprises the use of
laser capture microdissection (LCD) to dissect and purify taste
tissues from non-taste tissues and/or step (i) comprises RNA
amplification of genes from taste cells and lingual cells and the
amplified genes are screened against a gene chip containing a
sample of genes specific to the particular mammal from which the
taste and lingual tissues are obtained.
[0118] Further in these methods step (i) preferably comprises high
throughput PCR using primers for each ion channel in the human or
non-human primate genome and step (ii) is preferably effected by in
situ hybridization using antisense RNA probes specific for the
genes identified in step (i) to determine level of expression in
taste versus lingual cells or by use of immunocytochemical
detection using a labeled antibody specific to the protein encoded
by gene or genes identified in step (i).
[0119] Also more generally, it is an object of the invention to
provide a method for identifying a gene encoding a polypeptide
involved in salty taste perception in a primate (human or
non-human) comprising:
[0120] (i) identifying a set of genes including genes which are
expressed in fungiform, circumvallate, foliate, or palate taste
cells but which are not expressed in lingual cells and/or genes
which are expressed in said taste cells at substantially higher
levels than in lingual cells;
[0121] (ii) of the genes identified in (i) identifying a set of
genes which are not expressed in taste cells which express umami,
sweet, bitter, or sour taste receptors or markers of these cells
(T1Rs or T2Rs or TRPM5 or PKD2L1/PKD1L3);
[0122] (iii) of the genes identified in (i) or (ii) optionally
identifying whether the gene is specifically expressed in the top
half of taste buds and not the bottom half or is enriched
(expressed at least 1.2-1.5 fold higher) in the top half of taste
buds relative to expression in the bottom half of taste buds;
and
[0123] (iv) determining, in a primary neuron which expresses one or
more genes identified according to (ii), which of said genes
functions as a sodium responsive ion channel or sodium responsive
receptor or transporter and thereby identifying this gene or genes
as a putative gene that modulates salty taste.
[0124] (Again the selected taste specific gene which is
functionalized will preferably be enriched i.e., expressed 1.2-1.5
fold higher in the top half of taste buds versus the bottom half of
taste buds.)
[0125] Also more specifically, it is an object of the invention to
provide an assay for identifying a compound having potential in
vivo application for modulating human salty taste comprising the
following:
[0126] (i) contacting a cell that expresses a gene encoding a
TRPML3 ion channel alone or with NALCN, NKAIN3, TRPML1, or TRPML2
or an ortholog or a gene encoding a polypeptide possessing at least
90% sequence identity to the polypeptide encoded thereby with at
least one putative antagonist, agonist or enhancer compound;
[0127] (ii) assaying sodium conductance, receptor activity or
sodium transport in the presence and absence of said putative
agonist, antagonist or enhancer; and
[0128] (iii) identifying the compound as a potential salty taste
enhancer based on whether it modulates sodium conductance and other
conductance properties consistent with a human salt receptor.
[0129] Also more specifically, it is an object of the invention to
provide a method of using a probe specific to a TRPML3 gene or gene
product to identify and/or isolate and or enrich salty taste
specific cell, preferably primate alt receptor expressing cells, in
a taste cell sample.
[0130] Also more specifically, it is an object of the invention to
provide the use of TRPML3 to purify or enrich a desired taste cell
subtype or taste cell lineage that includes the use of a
fluorescence activated cell sorter (FACS) or the use of labeled
magnetic beads.
[0131] Also more specifically, it is an object of the invention to
provide the use of TRPML3 to purify or enrich a desired taste cell
subtype or taste cell lineage wherein the desired taste cell
subtype or taste cell lineage is isolated, purified, enriched or
marked by a method that includes a negative cell selection
technique that eliminates at least one non-target taste cell
subtype or lineage based on the expression or absence of expression
of at least one other taste cell specific gene., e.g., by the use
of cytotoxic antibodies that specifically kill at least one
non-target cell type or lineage.
[0132] Also more specifically, it is an object of the invention to
provide methods of identifying, isolating or enriching salty taste
receptor cells using TRPML3 alone or in association with other
taste specific genes such as TRPML1, TRPML2, NALCN and/or NKAIN3 as
a marker.
[0133] Based on the foregoing, it can be appreciated that this
invention was in part the result of a novel protocol for
identifying taste specific genes. These genes were identified using
two different techniques, gene chips and a polymerase chain
reaction (PCR) screen, to identify novel salt receptor target
genes. First, Affymetrix gene chips containing most all known
macaque genes are used to determine which genes are specifically
expressed in primate circumvallate at the back of the tongue and
fungiform papilla taste cells at the front of the tongue and not
lingual epithelial cells isolated by laser capture microdissection.
Second, PCR is used to determine which ion channels, from channels
we have cataloged in the human/macaque genomes, are specifically
expressed in macaque fungiform and/or circumvallate (CV) papilla
taste cells but not lingual epithelial cells isolated by laser
capture microdissection. In addition, of these genes a subset which
is expressed specifically in the top half of taste buds, or which
is enriched (expressed at least 1.2-1.5 fold higher) is identified
as being especially preferred candidates for functionalization.
Taste-specific expression of genes identified by either approach,
are confirmed using an independent histological method such as in
situ hybridization or immunohistochemistry, to determine which
genes are expressed in taste cells. Using double labeling
histological methods, it is determined what novel taste-specific
genes are expressed in sweet, bitter, and umami cells that express
the taste-specific ion channel TRPM5, sour cells that express the
taste-specific ion channel PKD2L1/PKD1L3, or a unique cell type
that does not express TRPM5 or PKD2L1/PKD1L3. A taste-specific
gene, preferably an ion channel, that is conductive or activated by
sodium and is expressed in a TRPM5- and PKD2L1/PKD1L3-negative cell
population is a probable candidate for screening efforts to
identify the gene(s) that encode mammalian salty taste receptors,
as well as specific cell types wherein these salty taste receptor
genes are expressed such as in the oral cavity and urinary tract,
and also for use in high throughput assays designed to identify
enhancers of saltiness in humans. Using these general methods
TRPML3 was identified as a potential salty taste receptor.
[0134] Novel taste-specific genes identified using these rationales
as well as affecting salt perception (and other biological
activities likely affected thereby such as sodium absorption,
transport and excretion and the effects thereof such as fluid
retention and blood pressure regulation) may alternatively affect
other taste modalities and flavor perception in general. While, the
inventors have identified TRPML3 as being a salty taste receptor
and have convincing functional data in support thereof it is
anticipated that TRPML3 is involved in non-taste biological
functions such as discussed above. Therefore, this gene is a useful
target in therapeutic screening assays, e.g., for identifying
therapeutics for the treatment of diseases related to TRPML3 such
as Addison's Disease, mocoid disorders such as mucolipidosis type
IV, urinary disorders, and cardiovascular disorders and pathologies
associated with sodium transport, metabolism, and excretion and
vasopressin or aldosterone release or production.
[0135] Also, the invention generally relates to use of the
inventive taste specific genes and probes specific thereto in
isolation and purification methods that include both positive and
negative cell separation methods. For example, desired taste cell
lineages or types may be isolated by positive cell selection
methods e.g., by the use of fluorescence activated cell sorting
(FACS), magnetic bead cell selection e.g., by visual identification
of desired cells such as individual transfected cells by
electrophysiology using antibody coated beads. Alternatively,
desired taste cell lineages or types may be recovered or purified
by negative cell purification and isolation methods wherein the
desired cell types are enriched or purified from a mixed cell
population by the removal of one or several undesired cell lineages
e.g., by contacting a mixed cell suspension containing the desired
taste (salty) cells and undesired cells e.g., derived from the
tongue, oral cavity or gastrointestinal tract and associated organs
with cytotoxic antibodies specific to a target gene or genes
expressed on the undesired taste cell type(s) which are to be
removed.
[0136] Also, the invention generally relates to use of the
inventive taste specific gene which is involved in specific taste
and non-taste specific functions, mapping of cell comprised on
specific regions of the gastrointestinal tract and associated
organs such as the intestinal epithelium or urinary tract that
express specific taste specific genes and which therefore are
involved in one or more of the taste cell specific functions
disclosed herein, and/or the use of the subject genes and markers
specific thereto in taste cell differentiation studies, e.g. for
identifying compounds that induce the differentiation or
dedifferentiation of taste cells e.g., adult or embryonic stem
cells and other pluripotent or immature cell types into desired
taste cell lineages and taste cell types.
[0137] Also more specifically, as described in detail infra, the
invention more broadly provides a rationale and criteria for a
candidate salty taste gene, preferably an ion channel which
are:
[0138] a) Specific expression in primate (macaque) taste cells,
particularly fungiform and/or circumvallate papilla derived taste
cells, but also foliate and palate taste cells, and not lingual
epithelial cells or expression at higher levels in taste cells than
lingual cells
[0139] b) Expression in a taste cell by histological methods.
Specifically, expression in a unique taste cell type that does not
express the sweet, bitter, and umami cell marker TRPM5 or the sour
cell marker PKD2L1/PKD1L3. This unique cell type will likely
correspond to unique taste cell lineage, e.g., a dedicated salt
sensing or fat sensing cell.
[0140] c) Functional expression as a sodium channel or a
sodium-activated receptor with basal, constitutive function (i.e. a
fraction of the channel population is open and passing sodium at
rest) in heterologous expression systems (such as Xenopus oocytes
and mammalian cells) or primary neurons (such as dorsal root
ganglia neurons).
[0141] d) Optionally, specific expression or enrichment in the top
fraction of taste bud cells, preferably at least 1.2-1.5 fold
higher expression in the top half versus bottom half of taste
buds.
[0142] Genes fulfilling these criteria are advanced into
high-throughput screening efforts to identify compounds that
enhance human salt perception. These methods coupled with in vitro
functional assays and neurophysiological data in mice expressing a
mutant TRPML3 gene that gives rise to the Varitint-waddler
phenotype have revealed that TRPML3 is a salty taste receptor in
primates (humans) and non-human primates and most likely other
animals including e.g., other mammals such as dogs, cats, horses,
bovines, pigs, sheep, and other vertebrates.
[0143] More specifically, as described in detail infra, the
invention provides a rationale and criteria for a candidate salty
taste gene, preferably an ion channel which are:
[0144] a) Specific expression in primate (macaque) taste cells,
particularly fungiform and/or circumvallate papilla derived taste
cells, but also foliate and palate taste cells, and not lingual
epithelial cells or expression at higher levels in taste cells than
lingual cells
[0145] b) Expression in a taste cell by histological methods.
Specifically, expression in a unique taste cell type that does not
express the sweet, bitter, and umami cell marker TRPM5 or the sour
cell marker PKD2L1/PKD1L3. This unique cell type could be a
dedicated salt sensing cell.
[0146] c) Functional expression as a sodium channel or a
sodium-activated receptor with basal, constitutive function (i.e. a
fraction of the channel population is open and passing sodium at
rest) in heterologous expression systems (such as Xenopus oocytes
and mammalian cells) or primary neurons (such as dorsal root
ganglia neurons).
SUMMARY OF THE INVENTION
[0147] Using a novel rationale for identifying taste specific genes
disclosed in earlier provisional patent applications incorporated
by reference herein and which are claimed in a related application
filed on even date as this application the present inventors have
identified a taste specific polypeptide that functions as a primate
(human) salty taste receptor polypeptide and which in all
likelihood in involved in other physiological functions involving
sodium transport, absorption and excretion such as urinary and
cardiac functions.
[0148] Particularly, the inventors have identified a gene,
Mucolipin 3 (MCOLN3) or TRPML3 as it is alternatively named that
encodes a multitransmembrane protein expressed in the top of the
taste buds, in the taste sensory cells, that conducts sodium.
Various lines of evidence convincingly demonstrate that this
polypeptide as a primate salty taste receptor polypeptide.
[0149] Specifically, this gene represents a salt receptor that by
itself and/or in association with other taste specific polypeptides
or ion channels (related family members) such as TRPML1, TRPML2,
NALCN or NKAIN3 which allows sensory taste cells in the tongue's
taste buds to detect sodium chloride (salt). In addition, because
this gene is highly expressed in the adrenal and pituitary glands,
it is reasonably anticipated to play an active role in the
regulation of sodium metabolism in the body. The evidence that
points to this gene being the human salt receptor includes at least
the following:
[0150] (1) Using the novel rationale for identifying putative taste
receptor genes it was determined by the inventors that MCOLN3 is
specifically expressed in the top of the taste buds, in a subset of
taste sensory cells that do not express TRPM5 (that is, they are
not sweet, bitter or umami), do not express PKD2L1 (that is, they
are not sour) and towards the taste pore.
[0151] (2) It is known that MCOLN3 is also expressed in sensory
cells of other organs, like the ear. It is therefore a
`professional` sensory gene.
[0152] (3) It is further known that MCOLN3 is strongly expressed in
the adrenal glands. These glands play a very important role in the
regulation of sodium metabolism in the body. MCOLN3 is therefore
likely to be a key molecule in the regulation of sodium metabolism
and may regulate the production of aldosterone by the adrenal
glands.
[0153] (4) Related to the foregoing it is also known that a human
autoimmune disease (Addison's) is characterized by the destruction
of the adrenal glands. One of the telltale symptoms of this disease
is salt craving. The latter is likely to result from the presence
of autoantibodies against MCOLN3, or a mutation in this gene that
disrupts the function of MCOLN3 in taste buds.
[0154] (5) It is also known that MCOLN3 or TRPML3 is highly
expressed in pituitary glands which produce vasopressin that is
involved in urine production, further substantiating the probable
role of this gene in sodium excretion and urinary function.
[0155] (6) As substantiated by the data contained in the
experimental examples infra, MCOLN3 conducts sodium in
electrophysiology studies and exhibits biochemical characteristics
predicted and consistent for a human salt receptor (detection of
K+, Li+ and amiloride insensitivity).
[0156] (7) Neurophysiological experiments (nerve recordings) using
sodium in the varitint-waddler mouse (having TRPML3 mutation)
indicate that the Varitint mouse is impaired in its response to
sodium (does not exhibit a robust salty taste response). In
addition, it has been confirmed that these same mice are ablated of
TRPML3 or MCOLN3 expressing taste cells (salty taste cells)
establishing further that the unique taste cell subset of TRPML3
expressing taste bud cells is functional, i.e., they are a
prerequisite for salty taste perception.
[0157] (8) Cell based assays using mammalian cells and amphibian
oocytes which express mutated TRPML3 polypeptides (mutation results
in the ion channel being fixed in the "open" orientation) have
identified TRPML3 enhancers and blockers which should enhance or
block salty taste in taste tests.
[0158] This discovery is very significant as the identification of
MCOLN3 or TRPML3 as a human and other primates and rodent salty
taste receptor (and presumably a salty taste receptor in other
mammals or vertebrates) allows for the design of screening assays
using cells transfected with this gene for the purpose of
identifying agonists, antagonists or enhancers (modulators) of the
function of this molecule. These compounds may be used as taste
modulators and also may be useful as therapeutic agents for
treating and modulating cardiac and urinary related functions and
conditions such as high or low blood pressure, stroke, heart
attack, arrhythmia, fluid retention, aberrant sodium and metabolism
and excretion of other ions.
[0159] As noted above, this gene was originally found using a whole
genome screening strategy aimed at identifying genes specifically
expressed in the top of the human taste buds. (See provisional
applications incorporated by reference herein which identify TRPML3
as being a taste specific gene in rodents and primates.) The
inventors had also earlier determined from previous experiments
that the top of the taste buds contain cells that over-express
known taste receptor genes and other taste specific genes including
the sodium ion channel TRPML3 which is similarly enriched in the
top half of taste buds. In contrast, the bottom of the taste buds
contains precursor cells of the sensory taste bud cells that reside
in the top portion of the taste bud. This database allowed the
inventors to identify many genes specifically expressed by the top
(sensory) cells of the taste buds including TRPML3.
[0160] It was further noted by the inventors in reviewing the taste
specific genes identified that one of these genes, MCOLN3 or TRPML3
had been previously reported to be responsible for the phenotype of
a mouse mutant called varitint-waddler that exhibits early-onset
hearing loss, vestibular defects, pigment abnormalities and
perinatal lethality (DiPalma et al., Mutations in Mcoln3 associated
with deafness and pigmentation defects in variant-waddler (Va)
mice. Proc. Natl. Acad. Sci. USA 99: 14994-14999; 2002). As noted
in the background of the invention, MCOLN3 or TRPML3 is expressed
in the hair cells and plasma membrane of stereocilia (in the ears)
and a mutation resulting in an ala 419 to pro substitution in the
fifth transmembrane domain had specifically been reported to result
is a hyperactive MCOLN3 that causes in the death of cells
expressing this molecule, such as the hair cells of the ear (hence
the deafness of the Va mouse) (Grimm C et al., Proc Natl. Acad.
Sci. USA 104:19583-8; 2007).
[0161] Based on the inventors' elucidation of TRPML3 as a salt
receptor in primates and likely other mammals it was further
predicted that this mouse would also exhibit salty taste
abnormalities (due to the abnormal MCOLN3 molecule and its effect
in the taste bud cells of the tongue which will likely impair salty
taste perception). In fact this has been conformed by the
inventors. The inventors have conducted neurophysiology studies
(described infra in the experimental examples) using mice which
express this mutant TRPML3 gene (varitint mouse) and have
confirmed, as hoped and anticipated that that these mice exhibit
impaired responses to salty taste stimuli (as evidenced by nerve
recording results in these mice stimulated with salty taste stimuli
at concentrations where a positive nerve recording response would
normally be observed). Using CT nerve recordings, Varitint waddler
mice were shown to exhibit a deficiency in the response to sodium
chloride. Specifically, Varitint waddler mice have a greatly
reduced benzamil-insensitive CT nerve response to sodium
chloride.
[0162] Also, the inventors have molecular and immunohistochemical
data which revealed that these same mice have taste buds which are
ablated of the TRPML3 expressing taste bud cells. This confirms the
inventors' supposition that this unique taste cell subset was
involved in detecting salty taste and a prerequisite for salty
taste perception.
[0163] Therefore, the inventors have in vivo evidence
substantiating a conclusion that the presence of a functional
TRPML3 ion channel in specific taste cells ("professional" salty
taste cell) is a necessary prerequisite for salty taste perception
in rodents and likely other mammals including most especially
humans and other primates.
[0164] In addition, because, the inventors have determined using
public databases that MCOLN3 is expressed strongly in the adrenal
and pituitary glands this is further supportive of the inventors'
discovery as well as suggesting other applications of the gene and
compounds that specifically detect or target this gene and/or
modulate its function. The fact that this gene is expressed in
adrenal and pituitary glands is a key observation because the
adrenal glands represent one of the main sodium metabolism
regulators of the body. These glands monitor salt levels of the
blood, and secrete aldosterone (a mineralocorticoid) that regulates
blood pressure and water and salt balance in the body by helping
the kidney retain sodium and excrete potassium. When aldosterone
production falls too low, the kidneys are not able to regulate salt
and water balance, causing blood volume and blood pressure to
drop.
[0165] Also, the pituitary glands produce vasopressin (AVP) a
hormone that involves sodium levels in the urine and plays a role
in sodium excretion through the urine. Particularly, one of the
most important roles of AVP is to regulate the body's retention of
water; it is released when the body is dehydrated and causes the
kidneys to conserve water, thus concentrating the urine, and
reducing urine volume. It also raises blood pressure by inducing
moderate vasoconstriction.
[0166] In addition AVP increases the permeability to water of the
distal convoluted tubules and collecting tubules in the nephrons of
kidneys and thus allows water reabsorption and excretion of a
smaller volume of concentrated urine--antidiuresis. This occurs
through insertion of additional water channels (Aquaporin-2) into
the apical membrane of the tubules/collecting duct epithelial
cells. The aquaporin allows water to pass out of the nephron (at
the distal convoluted tubules and the conducting tubules) and into
the cell, increasing the amount of water re-absorbed from the
filtrate.
[0167] AVP also increases permeability of the medullary portion of
the collecting duct to urea, allowing increased reabsorption of
urea into the medullary interstitium, down the concentration
gradient created from the removal of water in the cortical
collecting duct. Moreover, another renal role for AVP is that it
stimulates sodium reabsorption in the thick-ascending loop of
henle. Therefore, based on the inventors' discovery that TRPML3 is
involved in salty taste detection, it is not surprising that TRPML3
is expressed in 2 glands which produce polypeptides very
significantly involved in sodium transport and excretion and that
this gene, aside from being involved in salty taste perception
plays an active role in regulating other processes involving sodium
transport, absorption and excretion and in particular processes
regulated by vasopressin or aldosterone.
[0168] Therefore, based on these observations and the elucidation
of this gene as a salty taste receptor, MCOLN3 was further
identified by the inventors as being a key salt/sodium monitoring
molecule in the adrenal glands that controls the production of
aldosterone and/or in regulating vasopressin release by the
pituitary glands.
[0169] In the tongue, as is anticipated for a molecule identified
as being a taste receptor that detects salty taste stimuli, MCOLN3
or TRPML3 is expressed by a subset of taste sensory cells located
in the top of the taste buds which are responsible for detecting
salty taste. Therefore, this invention provides compelling proof of
the pivotal role of MCOLN3 or TRPML3 in detecting and regulating
salt in various tissues.
[0170] While MCOLN3 had previously been reported to be a sodium
conducting channel, there are numerous sodium ion channel
polypeptides and this channel had not been previously recognized as
being involved in salty taste perception or in regulating sodium
metabolism, excretion, transport or sodium related processes
involving vasopressin and aldosterone. Therefore, this invention
constitutes a new and unexpected discovery as it provides a new use
(salty taste receptor) for a known gene (MCOLN3). In the pituitary,
the inventors further anticipate based on their discoveries and
data contained herein substantiating the role of TRPML3 as a salty
taste receptor that MCOLN3 is further likely involved in the
regulation of vasopressin release. As mentioned, vasopressin is a
key regulator of urine production through its effects on the
kidneys. Importantly, vasopressin release from the posterior
pituitary is known to be regulated by NaCl concentration. This
protein is highly expressed in the pituitary glands. Therefore,
based on its expression in the pituitary, TRPML3 through its
probable effect on vasopressin release, likely regulates NaCl
metabolism in the body, through its effects on fluid retention,
NaCl sensing and concentration, and blood pressure.
[0171] Therefore, in one embodiment the invention identifies MCOLN3
or TRPML3 as a human salty taste receptor and based thereon
provides screening assays using cells transfected with this gene
for the purpose of identifying agonists, antagonists or enhancers
(modulators) of the function of this molecule which will modulate
salty taste and other taste related TRPML3 functions and non-taste
related functions such as those involving sodium excretion,
metabolism, and transport in different tissues and pathological
conditions relating to aberrant TRPML3 expression such as are
identified herein.
[0172] More specifically, in another embodiment the invention
provides an isolated and purified taste, adrenal, pituitary or
urinary organ cell or enriched taste cell sample comprising a
taste, adrenal, pituitary or urinary organ cell that expresses
TRPML3 that is involved in salty taste perception, sodium
metabolism, aldosterone production, and/or vasopressin release
wherein said isolated taste, adrenal, pituitary, or urinary organ
cell or enriched taste cell sample expresses TRPML3 gene or a
variant thereof that encodes a sodium channel that modulates at
least one of salty taste, sodium metabolism, aldosterone production
and vasopressin release. Preferably the cell will be of human,
non-human primate or rodent origin.
[0173] Also in another embodiment the invention provides an
isolated taste receptor that modulates salty taste perception
comprising a TRPML3 polypeptide or variant thereof that is useful
in assays for identifying TRPML3 modulators and/or which taste
receptor polypeptide modulates salty taste in mammals including
humans. This taste receptor may be monomeric or polymeric
(homopolymeric or heteropolymeric) and may comprise other taste
specific polypeptides, e.g., other ion channel polypeptides such as
NKAIN3 or NALCN or related ion channels such as TRPML1 or TRPML2.
This TRPML3 polypeptide or nucleic acid sequence may be of
mammalian or other species origin. As mentioned, given the
importance of sodium metabolism and excretion to organism's
homeostasis and well being, it is likely that this gene and its
various species orthologs play a role in salty taste perception and
salt (sodium) metabolism, and excretion in different mammals and
likely other vertebrates such as reptiles, amphibians and
avians.
[0174] The TRPML3 genes according to the invention may be wild-type
or may be genetically engineered to introduce desired mutations
that affect (enhance or inhibit) ion channel function and/or which
fix the ion channel in an open or closed orientation, also, it may
be modified by the substitution of host preferred codons. Such
mutations are exemplified herein and one skilled in the art will be
able to design others using methods known in the art. Therefore, it
should be understood that the TRPML3 polypeptides herein may be
modifies relative to the native TRPML3 polypeptide, and may possess
80, 85, 90, 95, 96, 97, 98, 99, or greater sequence identity to
native TRPML3 polypeptide or a functional fragment. In addition,
the subject TRPML3 polypeptides may comprise chimeric ion channels,
i.e., wherein one or more domains or regions of the endogenous
TRPML3 ion channel are substituted by the corresponding domain or
region of a related (e.g. an ortholog) ion channel, an ion channel
in the same TRPML family (TRPML1 or TRPML2) or another ion channel,
e.g., another sodium channel such as NALCN or NKAIN3.
[0175] These chimeras may be constructed based on the known TRPML3
protein topology. This topology is depicted schematically
below.
[0176] In this schematic the transmembrane domains are listed 1
through 6. The amino (N) and carboxy (C) are inside the cell. In
addition, there is a large extracellular loop between TM1 and TM2
that resides outside the cells. Chimeras that are functional (still
respond to sodium) can e.g., potentially be constructed by
substituting the extracellular loop region spanning TM1 and TM2
with that of another TRPML3 or another ion channel polypeptide or
by substituting a TM with the corresponding TM of another ion
channel. Also, chimeras can be made between human and mouse TRPML3
in which the large extracellular loop between TM1 and TM2 is
swapped.
[0177] Also, residues around the pore region and TM5 potentially
may be modified, e.g., by corresponding residues in other ion
channels.
[0178] Based on a comparison and alignment of the protein sequences
derived from human (NM.sub.--018298) and mouse (NM.sub.--134160)
TRPML3 sequences. (FIG. 35) wherein human is denoted Hs and mouse
is denoted Mm it can be seen that these proteins are 91% identical
and 96% similar. This substantiates the inventors' supposition
(reasonable) that TRPML3 likely is well conserved in different
mammals and potentially other vertebrates given the important
physiological functions it regulates and that chimeras and mutants
which are functional may be constructed. In fact, this application
contains sequences for avian and fish TRPML3 genes in the Sequence
Listing preceding the claims. Other orthologs may be identified
using these and other TRPML3 genes as probes.
[0179] The alignment in the figure (FIG. 35) similarly shows the
six transmembrane domains in the TRPML3 polypeptides for both human
and mouse TRPML3 are underlined TM1 through TM6. The pore region
between TM5 and TM6 is denoted `pore region`. As in the schematic
above, the amino and carboxy termini are predicted to be located
inside the cells. In constructing mutants it may be desirable to
retain the residues in the pore region intact or to modify very few
residues with these modifications if present corresponding to the
residues present in the pore region of other TRPML3 polypeptides or
to the corresponding residues in the pore region of TRPML1 or
TRPML2.
[0180] The A419P mutation discussed herein and found in the
varitint-waddler mouse locks TRPML3 in the open conformation and is
in TM5 and is highlighted in FIG. 35.
[0181] As discussed and shown in the examples infra, this mutation
is useful in assays for TRPML3 modulators (blockers) and in
particular may be used in FLIPR assays. Another mutation, V412P,
partially activates TRPML3 and is denoted in magenta. This mutation
increases TRPML3 activity and can be used to screen for enhancers
in a FLIPR assay. Also, other mutations potentially can be made
around TM5 and the pore region to alter TRPML3 ion channel activity
and generate active channels that could be used in high-throughput
screens.
[0182] In general, this invention uses assays that include the use
of a wild-type or mutated TRPML3 polypeptide or one wherein the
codons are optimized for the host cell wherein expression takes
place, e.g., a human cell. However, in some instances it is desired
to determine all of the effects of TRPML3 in vivo by eliminating
the expression of the TRPML3 polypeptide. Therefore, in another
embodiment the invention provides a transgenic non-human animal
which has been genetically engineered to knock out the expression
of endogenous TRPML3 or to express a non-functional TRPML3
polypeptide.
[0183] Also in another embodiment the invention provides a
transgenic non-human animal which has been genetically engineered
to express a heterologous TRPML3 polypeptide or a mutant or
chimeric TRPML3 polypeptide so that the animal may be screened to
identify modulators of this heterologous TRPML3 polypeptide, e.g.,
human or other primate TRPML3 or a TRPML3 of a domesticated animal
(dog, cat, etc.)
[0184] Also in another embodiment the invention provides assay
methods of using a transgenic animal that expresses a mutant or
heterologous TRPML3 in screens to identify antagonists, agonists or
enhancers of TRPML3 (so that the animal may be screened to identify
modulators of the mutant or heterologous TRPML3 polypeptide).
[0185] Also, in a related embodiment the invention produces
transgenic animals containing the TRPML3 mutation found in the
Varitint-mouse or other functionally equivalent mutations in order
to create transgenic animals depleted of specific types of TRPML3
expressing cells, such as salty taste cells, pituitary cells,
adrenal cells, melanocytes, or urinary organ system cells and to
study the effects of these cell ablations in these animals or to
use these animals as disease models for conditions involving these
ablated cells.
[0186] Also in another embodiment the invention provides methods of
using a transgenic animal according wherein the TRPML3 gene has
been "knocked out" in order to elucidate the effect of TRPML3 on
taste, and on cardiac or urinary function and in particular on
aldosterone production, sodium metabolism, salty taste perception
or vasopressin release.
[0187] Also, as the TRPML3 knock-out should give rise to conditions
or abnormalities involving aberrant sodium transport, absorption
and excretion and related urinary or cardiovascular effects these
transgenic animals potentially may be used as models for these
conditions and for testing potential therapeutics and therapeutic
regimens.
[0188] Also, in a more specific embodiment the invention provides
the use of use of Varitint waddler mice to study salty taste
behavior in the absence of TRPML3 taste cells.
[0189] Also, in a more specific embodiment the invention relates to
the use of the Varitint waddler mice to detect the effect of TRPML3
function on melanocytes, pituitary, adrenal, taste, urinary or
taste cells.
[0190] Also, in a more specific embodiment the invention relates to
the use of the Varitint waddler mice in assays to detect genes
specifically expressed in salty taste cells and not in the Varitint
waddler mice (as salty taste cells are ablated therein) which genes
may modulate TRPML3 function, or function as a salty taste receptor
or modulate transmission of salty taste signaling from TRPML3 to
the nerve fibers and/or control the development differentiation or
apoptosis of salty taste cells. These gene detection assays may
comprise the use of gene chips or microarray technology to compare
the genes expressed in salty taste cells versus genes expressed in
Varitint waddler mice.
[0191] Also, in a more specific embodiment the invention provides
methods of treating parathyroid related diseases such as calcium
homeostasis, hypercalcemia, osteitis, hypoparathyroidism,
hyperparathyroidism, osteitis fibrosis cystica,
pseudohypoparathyroidism, Jansen's metaphyseal chondroplasia,
Blomstrand's chondroplasia, and osteoporosis of different causes
such as diseases, age, menopause, chemotherapy, radiation therapy,
drugs and the like.
[0192] Also in another embodiment the invention provides a
recombinant cell which expresses a salty taste receptor comprising
TRPML3 or a variant thereof that encodes a sodium ion channel
polypeptide.
[0193] Also, in another embodiment the invention provides the use
of A419P TRPML3 polypeptide as a toxin to kill specific cell types,
e.g. salty taste cells, pituitary cells, adrenal cells,
melanocytes, and/or urinary organ system cells which express
TRPML3.
[0194] Also, in another embodiment the invention provides the use
of labeled molecules that specifically bind TRPML3 to study sodium
transport, metabolism, or excretion by the body.
[0195] Also, in another embodiment the invention provides the use
of molecules that specifically bind TRPML3 to direct therapeutics
or diagnostic agents to specific sites, e.g., salty taste cells,
adrenal cells, melanocytes, pituitary cells, et al.
[0196] Also more specifically, in another embodiment the invention
provides an assay for identifying compounds that agonize,
antagonize or enhance an activity of TRPML3 comprising contacting a
recombinant or endogenous taste or other cell that expresses TRPML3
with a putative TRPML3 enhancer, agonist or antagonist and
determining the effect thereof on TRPML3 activity. Preferably these
assays will be electrophysiological assays e.g., patch clamp or two
electrode voltage clamping assays.
[0197] Also more specifically, in another embodiment the invention
provides methods for identifying TRPML3 modulators by an ion flux
assay.
[0198] Also more specifically, in another embodiment the invention
provides such TRPML3 assays wherein the identified agonist,
antagonist, or enhancer compounds are evaluated in a taste
test.
[0199] Also more specifically, in another embodiment the invention
provides such TRPML3 assays wherein the effect of the identified
agonist, antagonist, or enhancer compounds on aldosterone
production is tested in an animal.
[0200] Also more specifically, in another embodiment the invention
provides such TRPML3 assays wherein the effect of the identified
agonist, antagonist, or enhancer compounds on vasopressin release
is tested in an animal.
[0201] Also more specifically, in another embodiment the invention
provides such TRPML3 assays wherein the effect of the identified
agonist, antagonist, or enhancer compounds on at least one of
cardiac or urinary function and more specifically on blood
pressure, fluid retention, sodium metabolism or urine production,
wherein this is tested in an animal.
[0202] Also more specifically, in another embodiment the invention
provides the use of the identified agonist, antagonist, or enhancer
compounds for treating a disease or condition involving aldosterone
production comprising administering an effective amount of a
compound that modulates TRPML3.
[0203] Also more specifically, in another embodiment the invention
provides the use of the identified agonist, antagonist, or enhancer
compounds for treating a disease or condition involving vasopressin
release comprising administering an effective amount of a compound
that modulates TRPML3. As mentioned, these conditions include by
way of example diseases and conditions treatable using TRPML3
modulators which agonize or antagonize vasopressin such as
diabetes, obesity, kidney diseases such as cystic kidney disease,
acquired renal cystic disease, ocular circulation related disorders
such as myopia; nausea, emesis, sexual dysfunction (male or
female), edema, hypertension, congestive heart failure (ranging
from class II of the New York Heart Association to florid pulmonary
edema), periodic idiopathic edema, nephrotic syndrome, ascites due
to cirrhosis or other causes, cerebral edema of various causes, as
well as dilutional hyponatremia and metabolic alterations
collectively known as the syndrome of inappropriate ADH secretion
and other diseases or conditions wherein vasodilation and/or
antioxytocic activity is therapeutically desirable.
[0204] Also more specifically, in another embodiment the invention
provides the use of the identified agonist, antagonist, or enhancer
compounds for treating a disease or condition involving aldosterone
production comprising administering an effective amount of a
compound that modulates TRPML3 and thereby aldosterone. Diseases
and conditions treatable using TRPML3 modulators which agonize or
antagonize aldosterone and thereby sodium transport and excretion
include by way of example edema, blood pressure (hyper or
hypotension), liver cirrhosis, primary hyperaldosteronemia, renal
dysfunction, diabetes (Type I or II) and the pathological symptoms
associated therewith including circulatory problems, edema, ocular
disorders relating to poor circulation, hypercortisolaemia,
atherosclerosis or obesity, e.g., abdominal obesity, as well as
liver disease, sexual dysfunction (male or female), cerebrovascular
disease, vascular disease, retinopathy, neuropathy, insulinopathy,
endothelial dysfunction, baroreceptor dysfunction, migraine
headaches, hot flashes, and premenstrual tension and other
cardiovascular conditions such as atherosclerosis, heart failure,
congestive heart failure, vascular disease, stroke, myocardial
infarction, endothelial dysfunction, ventricular hypertrophy, renal
dysfunction, target-organ damage, thrombosis, cardiac arrhythmia,
plaque rupture and aneurysm.
[0205] Also more specifically, in another embodiment the invention
provides the use of the identified agonist, antagonist, or enhancer
compounds for treating a disease or condition involving TRPML3 such
as Addison's disease, or type IV mucolipidosis.
[0206] Also more specifically, in another embodiment the invention
provides the use of the identified agonist, antagonist, or enhancer
compounds for modulating cardiac function, e.g., blood pressure,
arrhythmia, or stroke or fluid retention in a subject in need
thereof comprising administering an effective amount of a compound
that modulates TRPML3.
[0207] Also more specifically, in another embodiment the invention
provides the use of such identified agonist, antagonist, or
enhancer compounds for modulating urine production and/or excretion
in a subject in need thereof comprising administering an effective
amount of a compound that modulates TRPML3.
[0208] Also more specifically it is another embodiment to provide
the use of TRPML3 modulators, e.g., enhancers, agonists or
antagonists, for treating conditions involving melanocytes such as
melanoma and pigmentation disorders and to promote the growth and
coloration of hair or skin which has lost coloration, e.g. because
of disease, aging, UV radiation, chemotherapy, or hormone
imbalance.
[0209] Also more specifically it is another embodiment to provide
the use of TRPML3 modulators, e.g., enhancers, agonists or
antagonists, for treating conditions involving pituitary cells such
as pituitary cancer or diabetes or pituitary diseases.
[0210] Also more specifically it is another embodiment to provide
the use of TRPML3 modulators, e.g., enhancers, agonists or
antagonists, for treating conditions involving adrenal cells such
as adrenal cancer or other adrenal conditions.
[0211] Also more specifically it is another embodiment to provide
the use of TRPML3 modulators, e.g., enhancers, agonists or
antagonists, for treating conditions involving taste buds such as
taste bud related malignancies other taste bud related
conditions.
[0212] Also more specifically, in another embodiment the invention
provides such identified agonist, antagonist, or enhancer compounds
useful in taste and therapeutic applications which may include
polypeptides, antibodies, small molecules, siRNAs, antisense RNAs,
ribozymes et al.
[0213] The discovery of TRPML3 as a salty taste receptor was based
in part on the hypothesis that human salty taste may be mediated,
in part, by a sodium or other ion channels as well as transporters
and GPCRs expressed specifically in taste cells. The compounds
identified using these gene products and their derivatives that
modulate the activity of these target genes potentially can be used
as modulators of human salty taste in foods, beverages and
medicinals for human consumption. Also, such compounds and their
derivatives potentially may be used to treat diseases involving
aberrant ion channel function. Further the compounds identified
using genes identified according to the invention and cells which
express same are useful in therapeutic screening assays as
discussed herein for identifying potential therapeutics that
modulate other taste-cell related functions and phenotypes.
[0214] This gene was deemed significant by the inventors based on
its selective expression in primate fungiform papilla taste cells
found at the front of the tongue and circumvallate papilla taste
cells found at the back of the tongue using gene-chips microarrays
from taste receptor cells as compared to non-taste lingual
epithelial cells isolated by laser capture microdissection (LCM).
This protocol also identified 2 other taste specific ion channels
NKAIN3 and NALCN which are enriched in the top half of the taste
buds. Since salt perception is most prevalent at the front of the
tongue, a salt receptor gene was predicted to be contained within
this set of identified genes. (It is stated throughout the
application that the inventors have identified "a human or
mammalian salty taste receptor" rather than "the human or mammalian
salty taste receptor" since it is conceivable that humans or other
mammals may have some redundancy in the genes that regulate salty
taste and sodium metabolism.
[0215] The subject gene was initially identified as being a taste
specific ion channel polypeptide putatively involved in salty taste
in mammals. This protocol involved the steps of (i) identifying a
set of genes including genes which are expressed in macaque taste
(fungiform and circumvallate papilla taste cells) but which are not
expressed in lingual epithelial cells and/or genes which are
expressed in taste cells at substantially higher levels than in
lingual cells; (ii) identifying a subset of genes within the set of
genes identified in (i) which are selected based on criteria which
suggest that they are likely salt receptor candidates, i.e.,
putative ion channels and/or encode multidomain transmembrane
proteins. These genes were then examined to determine whether these
genes are expressed or not expressed in taste cells which express
umami, sweet or bitter taste receptors (T1Rs or T2Rs) or sour taste
receptors (PKD2L1/PKD1L3); and (iii) functionally expressing one or
more genes in the subset identified according to (ii) and
determining which of these genes function as a sodium responsive
ion channel or sodium responsive receptor or transporter and
thereby identifying this gene or genes as a putative gene that
modulates salty taste. Typically, the taste tissues for this method
are derived from human, primate, or rodent sources. In one
preferred embodiment of the method, the genes in step (iii)
function as sodium responsive ion channels, and more preferably,
when the genes are expressed, a fraction of the channel population
is open and passing sodium at rest.
[0216] In a preferred embodiment, step (i) comprises the use of
laser capture microdissection (LCM) to dissect and purify taste
tissues from non-taste tissues. In one mode of this embodiment,
step (i) comprises RNA amplification of genes from taste cells and
lingual cells and the amplified genes are screened against a gene
chip containing a sample of genes specific to the particular mammal
from which the taste and lingual tissues are obtained, and
preferably, the gene chips include a set of annotated human genes.
In an alternative mode of this embodiment, step (i) comprises high
throughput PCR using primers for each ion channel in a mammalian
genome.
[0217] In another preferred embodiment, step (ii) is affected by in
situ hybridization using antisense RNA probes specific for the set
of genes identified in step (i) to determine level of expression in
taste versus lingual cells. In an alternative preferred embodiment,
step (ii) is affected by use of immunochemical detection using a
labeled antibody specific to the protein encoded by gene or genes
identified in step (i).
[0218] In another embodiment of the method for identifying a gene
encoding a polypeptide involved in salty taste perception in a
mammal, the method of this invention comprises the steps of (i)
identifying a set of macaque genes including genes which are
expressed in taste cells but which are not expressed in lingual
cells and/or genes which are expressed in taste cells at
substantially higher levels than in macaque lingual cells; (ii)
identifying a subset of genes within the set of genes identified in
(i) which are not expressed in taste cells which express umami,
sweet or bitter taste receptors (T1Rs or T2Rs) or sour taste
receptors (PKD2L1/PKD1L3); and (iii) determining, in a primary
neuron which expresses one or more genes in the subset identified
according to (ii), which of said genes functions as a sodium
responsive ion channel or sodium responsive receptor or transporter
and thereby identifying this gene or genes as a putative gene that
modulates salty taste. In one mode of this embodiment, step (iii)
comprises contacting the neuron with an antibody which specifically
binds the gene and inhibits its function.
[0219] In another generic mode, this invention provides an assay
for identifying a compound having potential in vivo application for
modulating human salty taste. This method comprises the steps of
(i) contacting a cell that expresses a gene encoding an ion
channel, receptor or transporter identified as a putative salty
taste affecting gene according to any one of the methods above, or
a gene encoding a polypeptide possessing at least 90% sequence
identity to the polypeptide encoded thereby, with at least one
putative enhancer compound; (ii) assaying sodium conductance,
receptor activity or sodium transport in the presence and absence
of said putative enhancer; and (iii) identifying the compound as a
potential salty taste enhancer based on whether it increases sodium
conductance, the activity of said receptor or sodium transport. In
various embodiments, the gene encodes an ion channel or the gene
encodes a GPCR. Preferably, the gene is a human gene. More
preferably, the method further includes testing the effect of the
compound or a derivative thereof in a human or animal taste test.
Preferably, the selected compound promotes sodium ion transport
into taste bud cells. The putative salty taste affecting gene may
be expressed in an amphibian oocyte, or in a mammalian cell,
preferably a Xenopus oocyte or a mammalian cell selected from the
group consisting of a HEK293, HEK293T, Swiss3T3, CHO, BHK, NIH3T3,
monkey L cell, African green monkey kidney cell, Ltk-cell and COS
cell. Preferably, the putative salty taste affecting gene is
expressed under the control of a regulatable promoter. A putative
salty taste affecting gene may be expressed stably or transiently.
In a preferred mode, the salty taste affecting gene is TRPML3.
[0220] In a preferred mode, the assay of step (ii) is an
electrophysiological assay which uses a sodium sensitive dye, and
preferred dyes include membrane potential dyes selected from the
group consisting of Molecular Devices Membrane Potential Kit
(Cat#R8034), Di-4-ANEPPS (pyridinium,
4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)-1-(3-sulfopropyl)hydroxid-
e, inner salt, DiSBACC4(2)(bis-(1,2-dibabituric acid)-triethine
oxanol), Cc-2-DMPE (Pacific Blue
1,2-dietradecanoyl-sn-glycerol-3phosphoethanolamine,
triethylammonium salt) and SBFI-AM (1,3-benzenedicrboxylic acid,
4,4-[1,4,10-trioxa-7,13-diazacylopentadecane-7,13-diylbis(5-methoxy-6,1,2-
-benzofurandiyl)}bis-tetrakis{(acetyloxy)methyl}ester (Molecular
Probes), more preferably, the sodium sensitive dye is sodium green
tetraacetate (Molecular Probes) or Na-sensitive Dye Kit (Molecular
Devices). In another preferred mode, the assay of step (ii) is a
two electrode voltage clamping assay in Xenopus oocytes, or the
assay is a patch clamp assay in mammalian cells. Preferably, the
assay measures activity by an ion flux assay, including using
atomic absorption spectroscopy to detect ion flux.
[0221] Alternatively, the assay may use a fluorescence plate reader
(FLIPR), or a voltage imaging plate reader (VIPR), which is used to
increase ion channel-dependent sodium or fluid absorption. In a
preferred embodiment of this method, the activity of the putative
salty taste affecting gene is assayed in a frog oocyte
electrophysiologically by two electrode voltage clamping, or in a
mammalian cell, preferably using an automatic imaging instrument,
which may be a fluorescence plate reader (FLIPR) or a voltage
imaging plate reader (VIPR) or patch-clamping.
[0222] In yet another mode, this invention provides an assay for
identifying a compound having potential in vivo application for
modulating human sweet, bitter, umami, or sour taste. This method
comprises the steps of (i) contacting a cell that expresses a gene
identified according to the invention with at least one putative
enhancer or blocker compound; (ii) assaying sodium conductance,
receptor activity or taste gene product function in the presence
and absence of said putative enhancer or blocker; and (iii)
identifying the compound as a potential enhancer or blocker for
sweet, bitter or umami taste based on whether it modulates sodium
conductance, the activity of said receptor or taste gene product
function.
[0223] In yet another mode, this invention provides an assay for
identifying a compound having potential in vivo application for as
a potential therapeutic. This method comprises the steps of (i)
contacting a cell that expresses a gene identified according to the
invention with at least one putative enhancer or blocker compound;
(ii) assaying sodium conductance, receptor activity or taste gene
product function in the presence and absence of said putative
enhancer or blocker; and (iii) identifying the compound as a
potential therapeutic that may be used to modulate a taste cell
related function or phenotype that does not directly involve taste
such a digestive disorder or disease, taste cell or taste bud
turnover or regeneration, immune regulation of the oral or
digestive system, or treatment of a metabolic disorder such as
diabetes, obesity, eating disorder et al., based on whether it
modulates sodium conductance, the activity of said receptor or
taste gene product function.
DETAILED DESCRIPTION OF THE FIGURES
[0224] FIG. 1 contains RT-PCR data that reveal that TRPML3 is a
taste-specific gene. In this experiment RT-PCR was affected using
human left) and monkey (right) taste buds (taste) and lingual
epithelial cells (lingual) samples collected by laser capture
microdissection. It can be seen that TRPML3 is only expressed in
taste cells, similar to the known taste-specific genes T1R2 and
TRPM5. The housekeeping gene beta-actin is expressed in both taste
and lingual cells demonstrating that RNA from both samples is of
high quality. `+` indicates reverse transcription was performed and
`-` indicates that no reverse transcription was performed (negative
control). Bands are only observed with reverse transcription. All
bands were cloned and sequenced to confirm gene identities.
[0225] FIG. 2 contain electrophysiological experiments which show
that TRPML3 forms a sodium channel. Whole cell patch clamp
electrophysiology of cells expressing human TRPML3 was affected.
The results of these experiments reveal that TRPML3 generates a
sodium leak current that is blocked upon removal of sodium and
replacement with the large impermeant cation NMDG. The top trace in
FIG. 2 shows current at a holding potential of -60 mV. The middle
traces in the Figure show current-voltage traces from -100 mV to
+60 mV in the presence (NaCl) and absence (NMDG-Cl) of sodium. The
bottom graph in FIG. 2 shows current voltage curves in the presence
(dark blue line; diamonds) and absence (magenta line; squares) of
sodium. It can be seen that TRPML3 exhibits inward rectification
(more current at negative voltages compared to positive
voltages)
[0226] FIG. 3 contains the results of other electrophysiology
experiments which indicate that human TRPML3 channel properties are
consistent with human salty taste psychophysics. The top graph in
FIG. 3 contains current-voltage curves showing TRPML3 sodium
conductance (dark blue line; diamonds) is not blocked by 30 uM
amiloride (magenta line; squares). Both human salty taste and
TRPML3 are not blocked by amiloride. The bottom graph in the same
Figure contains current-voltage curves showing TRPML3 is equally
permeable to the salty cations sodium (dark blue line; diamonds)
and lithium (magenta line; squares). This result is consistent with
TRPL3 being a human salty taste receptor given the fact that sodium
and lithium are known to be equally salty to humans since both
cations equally permeate the human TRPML3 channel.
[0227] FIG. 4 contains an experiment which shows that the TRPML3
protein is expressed in the apical membrane region near the taste
pore. It can be seen that the TRPML3 antibody labels taste cell
processes extending to the taste pore (left image). Magnification
of the apical taste bud domain facing the saliva clearly
demonstrates that TRPML3 protein is expressed at the taste pore
region (3 right images; taste pore denoted with blue arrows). This
observation is also consistent with TRPML3 being the human salty
taste receptor since this location is ideally suited for TRPML3 to
sense sodium in the saliva. Similar to TRPML3, other known taste
receptors (sweet, bitter, umami, and sour) are also polarized to
the taste pore where they sample saliva for their requisite
tastants.
[0228] FIG. 5 contains the data of an immunochemistry double
labeling experiment which indicates that the TRPML3 protein is not
expressed in TRPM5 cells. The figure shows the results of double
label immunohistochemistry with TRPM5 (green; left images) and
TRPML3 (red; middle images) in monkey CV papilla. It can be seen in
the Figure that cells expressing TRPM5 and TRPML3 are distinct
(merged images on the right). These data indicate that TRPML3 is
not expressed in TRPM5 cells (encompassing sweet, bitter, and umami
cells) but exclusively in professional salty taste cells.
[0229] FIG. 6 contains the data of another immunochemistry double
labeling experiment. The data contained in FIG. 6 reveal that the
TRPML3 protein is not expressed in PKD2L1 cells. The Figure
contains the results of a double label immunohistochemistry with
PKD2L1 (green; left images) and TRPML3 (red; middle images) in
monkey CV papilla. It can be seen from the Figure that cells
expressing PKD2L1 and TRPML3 are distinct (merged images on the
right). These data indicate that TRPML3 is not expressed in PKD2L1
cells (encompassing sour cells) but in professional salty taste
cells.
[0230] FIG. 7 illustrates an example of I/V curves in oocytes
injected with human TRPML3 cRNA.
[0231] FIG. 8 illustrates an example of screening oocytes with
human TRPML3 cRNA for compounds that may modulate TRPML3
activity.
[0232] FIG. 9 illustrates an example of I/V curves with the TRPML3
blocker gadolinium.
[0233] FIG. 10 is a flowchart of experiments used to examine the
effect of compounds in human TRPML3 activity in the oocyte
expression system using the two-electrode voltage clamp.
[0234] FIG. 11 illustrates the expression of constitutively active
sodium channels increase basal fluorescence in cells loaded with
specific membrane potential dyes.
[0235] FIG. 12 illustrates the application of gadolinium reduces
the increase in basal fluorescence in cells expressing
A419P-TRPML3.
[0236] FIG. 13 illustrates the application of gadolinium reduces
the increase in basal fluorescence in cells expressing A419P-TRPML3
in a dose-dependent fashion.
[0237] FIG. 14 illustrates the titration of TRPML3 plasmid.
[0238] FIG. 15 illustrates the effect of gadolinium is specific for
TRPML3.
[0239] FIG. 16 illustrates transducing HEK293 cells with
baculovirus encoding A419P-TRPML3 doubles the assay window.
[0240] FIG. 17 is an example of screening data obtained with
A419P-TRPML3 expressing cells.
[0241] FIG. 18 is a summary of a 10,000 compound miniscreen with
A419P-TRPML3 expressing cells.
[0242] FIG. 19 shows the alignment of wild-type (non-codon
optimized) and codon-optimized DNA sequence of human TRPML3 and
that these DNA sequences are 76.4% identical. Wild-type (non-codon
optimized), codon-optimized, and A419P TRPML3 were expressed in
oocytes and sodium currents were measured.
[0243] FIG. 20: Functional expression of human wild-type (non-codon
optimized; labeled WT with blue diamonds), codon-optimized (labeled
WT-CO with pink squares), and A419P (labeled mutant with yellow
triangles) TRPML3 cRNA. The inwardly rectifying I/V curves, denoted
by more current at hyperpolarized potentials (more negative
potentials) and less current at depolarized potentials (more
positive potentials), indicate functional expression of TRPML3 ion
channels. Note the augmented currents observed with codon-optimized
TRPML3 and A419P TRPML3 compared to wild-type TRPML3 with no codon
optimization
[0244] FIG. 21: Example of screening oocytes injected with
codon-optimized human TRPML3 cRNA to identify a compound (TRPML3
enhancer) that activates TRPML3. In multiple oocytes, the TRPML3
enhancer increased TRPML3 activity by 169+/-26% from
(representative trace on top) and had no effect on uninjected
oocytes with no TRPML3 expression (representative trace on bottom).
Addition of buffer only had no effect on TRPML3 currents and the
effects of the TRPML3 enhancer were reproducible upon a second
application.
[0245] FIG. 22: Example of TRPML3 enhancer effect on TRPML3 I/V
curve. Oocytes injected with codon-optimized human TRPML3 cRNA were
untreated (blue triangles labeled control) or stimulated with
TRPML3 enhancer (magenta squares labeled enhancer) and currents
were measured at voltages from -90 to +30 mV. Note that the TRPML3
enhancer activates TRPML3 current at negative voltages (inward
currents are larger with enhancer compared to with control),
resulting in an increase in the slope of the I/V curve. Note also
that the zero current shifts to the right, indicating an increased
sodium conductance in the presence of the enhancer
[0246] FIG. 23: Example of TRPML3 enhancer effect in the presence
and absence of extracellular sodium. Oocytes expressing
codon-optimized human TRPML3 cRNA were stimulated with NMDG (no
sodium), TRPML3 enhancer plus sodium, buffer only, or TRPML3
enhancer plus NMDG (no sodium). Note that TRPML3 enhancer increased
TRPML3 activity in the presence of sodium but had no effect in the
absence of sodium. These data demonstrate that the TRPML3 enhancer
opens TRPML3 channels and increases the flow of sodium ions into
the oocyte.
[0247] FIG. 24: Expression level of WT TRPML3 depends on the
mammalian cell type. A. Current voltage analysis (I/V plot) of
cells expressing WT and the A419P mutant TRPML3 channel in HEK293
cells. A419P mutant TRPML3 channels express large inward rectifying
currents (pink), whereas only small WT TRPML3 currents are observed
(blue). B. WT and A419P mutant TRPML3 channels have similar
functional characteristics in CHO cells.
[0248] FIG. 25: Use of TRPML3 for enhancer and blocker screening in
CHO cells. A. WT human TRPML3 channels transiently expressed in CHO
cells are used to identify channel enhancers. I/V plot shows that
compared to buffer alone (blue; control), use of the enhancer
results in an increase in inward current at negative potentials
(pink). B. Mutant A419P TRPML3 channel stably expressed in CHO
cells are used to identify channel blockers. Compared to buffer
alone (blue; control) addition of 1 mM GdCl.sub.3 (gadolinium
chloride) results in a decrease in inward current (pink).
[0249] FIG. 26: Use of codon optimized WT TRPML3 for the screening
of compounds which enhance TRPML3 function. A. Transient expression
of non codon optimized WT TRPML3 (light blue) results in little
current in HEK293 cells. In contrast, use of codon optimized WT
TRPML3 (Dark Blue; Cod Opt WT) results in currents with similar
average amplitude as A419P mutant channel (pink). B. Use of codon
optimized WT TRPML3 (blue) delivered with Baculovirus transduction
results in similar average currents as A419P TRPML3 (pink). C.
Cells transduced with codon optimized WT TRPML3 baculovirus is used
to identify enhancers of TRPML3 function. Compared to buffer alone
(blue; control) addition of enhancer compound results in an
increase in inward current (pink).
[0250] FIG. 27: Coexpression of WT and A419P TRPML3 increases
functional surface expression in HEK293 cells. A. Currents elicited
from A419P TRPML3 cDNA (0.5 ug) transfected into HEK293 cells,
yielding currents with characteristic inward rectification. B. WT
non codon optimized TRPML3 (1.5 ug) is expressed in HEK293 cells
and yields no currents. C. Coexpression of A419P (0.5 ug) with WT
(1.5 ug) TRPML3 cDNAs in HEK293 cells result in large inward
currents which are twice as large as those when expressing A419P
cDNA alone. D. I/V plot of the average currents elicited from WT
(blue), A419P (pink) and coexpression of WT and A419P (yellow)
TRPML3 cDNAs in HEK293 cells.
[0251] FIG. 28 contains an example of TRPML3 function in IonWorks
PPC patch plate. A, View of all 384 wells from a PPC patch plate
with an A419 TRPML3 inducible clone showing the results of the
pre-compound scan. Yellow indicates wells where the current at -120
mV was .ltoreq.0 nA (in control experiments with parental CHO-K1
cells none of the wells were labeled yellow). Blue indicates wells
were the average seal was too low (<10 mOhm) to measure the
current reliably. A419P TRPML3 currents could be measured in 94% of
the wells. B, Average currents .+-.SEM before and after addition of
4 mM GdCl3 or extracellular buffer (mock addition) from the patch
plate shown in A. GdCl3 was added to column 1-38 while
extracellular buffer was added to columns 39-48. For comparison,
data is included from a separate experiment with parental CHO-K1
cells. The stability of the TRPML3 current after mock addition
indicates that the assay should detect compounds that either
enhance or block TRPML3 currents.
[0252] FIG. 29 contains an example of an IonWorks scan with an
inducible CHO-K1 cell line expressing A419P TRPML3 (top panel).
TRPML3 inwardly rectifies, denoted by more current at
hyperpolarized potentials (more negative potentials) and less
current at depolarized potentials (more positive potentials).
Addition of GdCl3 blocks TRPML3 current. Red line denotes scan in
sodium (NaCl) solution. Blue line denotes scan in 4 mM GdCl3
solution. The middle panel is from parental CHO-K1 cells used as a
negative control. The positive currents at negative potentials are
due to leak subtraction overcorrecting the current at negative
potentials. The bottom panel show the voltage command protocol used
to record currents. The step from 0 mV to 10 mV is used to
calculate the leak current (current flowing through leaks in the
seal) which is subtracted from the total current to obtain the
current flowing through the membrane. Results are from single wells
in a PPC patch plate and represent the average current of up to 64
cells.
[0253] FIG. 30 contains a flowchart of experiments used to examine
the effect of compounds on human TRPML3 (hTRPML3) activity in the
IonWorks assay.
[0254] FIG. 31 shows that TRPML3 cells are specifically ablated
from taste buds in Varitint waddler mice. End-point RT-PCR
experiments on taste buds (TB) and lingual epithelial cells (LE) of
Varitint waddler (Va) or wild-type (WT) mice isolated by
laser-capture microdissection. TRPML3 is only expressed in taste
buds of WT mice and absent in taste buds of Va mice, whereas all
other taste genes (T1R2, GPR113, TRPM5) as well as housekeeping
genes (beta-actin, GAPDH) are equally expressed in TB and LE. `+`
indicates that reverse transcription was performed and `-`
indicates that no reverse transcription was performed. PCR bands
were only observed with reverse transcriptase indicating that PCR
products are derived from mRNA and not genomic DNA.
[0255] FIG. 32 also shows by use of real-time PCR that TRPML3 cells
are specifically ablated from taste buds in Varitint waddler mice.
Real-time quantitative RT-PCR experiments on taste buds of Varitint
waddler (Va) or wild-type (WI) mice isolated by laser-capture
microdissection. TRPML3 is only expressed in taste buds of WT mice
and absent in taste buds of Va mice (similar results were obtained
using two different primer sets labeled Mcoln3.sub.--1 and
Mcoln3.sub.--2), whereas all other taste genes (Tas1r2, Tas1r3,
PKD2l1, TRPM5, Plcb2, Tas2r108, and Tas2r116) as well as a
housekeeping gene (control) are expressed in taste buds from Va and
WT mice.
[0256] FIG. 33 contains an experiment showing that sweet, umami,
bitter and sour cells are intact in the taste buds of the
Varitint-waddler mouse. In situ hybridization of circumvallate
papilla from the back of the tongue of wild-type (top row of
images) and Varitint waddler (Va; bottom row of images) mice.
PKD1L3 (left; sour), PKD2L1 (middle; sour), and TRPM5 (right;
sweet, bitter, umami, and GPR113) taste cells were present at
similar levels in wild-type and Va mice.
[0257] FIG. 34 contains a CT nerve recording experiment
demonstrating that the Varitint waddler mice are deficient in salty
taste. CT nerve recordings from wild-type (left) or Varitint
waddler (Va; right) mice. Anterior tongues were stimulated with 0.1
M NaCl or 0.1 M NaCl plus 5 uM benzamil to inhibit the
amiloride-sensitive component of the CT nerve response. Tongues
were rinsed with a low salt solution containing 10 mM KCl in
between NaCl stimulations. Note that the benzamil-insensitive
component of the CT nerve response is largely eliminated in the Va
mouse (red arrows), indicating that ablation of TRPML3 taste cells
significantly impairs salty taste perception. In addition, the
immediate phasic response to NaCl is greatly reduced in the Va
mouse (red circles). Scale bars indicate time frames of salt
application (x-axis) and the magnitude of the CT response (y-axis;
arbitrary units).
[0258] FIG. 35 contains an alignment of the sequences of human and
mouse TRPML3 genes and polypeptides. The transmembrane domains,
extracellular loop and pore regions are identified as well as the
residue (419) that gives rise to the Varitint waddler mouse.
DETAILED DESCRIPTION OF THE INVENTION
[0259] The present invention relates to the identification of a
gene that regulates salty taste perception in mammals and
potentially other vertebrates, e.g., avians, reptiles and
amphibians. This gene, TRPML3 or MCOLN3 is specifically expressed
in taste cells that respond to salty taste stimuli and is also
expressed in pituitary, adrenal and melanocytes. This gene encodes
an ion channel polypeptide that alone or potentially in association
with other accessory molecules or ion channels such as TRPML1
TRPML2, NALCN or NKAIN3 detects salty taste stimuli and likely
regulates sodium transport, metabolism and excretion and/or further
may affect sodium related processes involving aldosterone and/or
vasopressin based on the fact that this ion channel is
substantially expressed in the adrenal gland which produces
aldosterone, a hormone significantly involved in sodium related
processes that affect the urinary and cardiovascular system as well
as other organs as well as being substantially expressed by the
pituitary, which secretes vasopressin, another hormone which plays
a very important role in sodium transport, metabolism and excretion
and which affects among other things blood pressure, urine output
and fluid retention. Therefore, the subject ion channel polypeptide
likely plays a significant role in sodium transport, metabolism and
excretion by different cells and organs, as well as being involved
in salty taste perception.
[0260] This gene, Mucolipin 3 (MCOLN3), or TRPML3 (identified using
a novel rationale disclosed herein and in other provisional
applications incorporated by reference herein) encodes a
multitransmembrane protein expressed in the top of the taste buds,
in the taste sensory cells, that conducts sodium. This gene is
believed to encode a salt receptor that allows sensory taste cells
in the tongue's taste buds to detect sodium chloride (salt). In
addition, because this gene is expressed in the adrenal and
pituitary glands, it may participate in the regulation of sodium
metabolism in the body. The evidence obtained by the inventors and
earlier reports relating to this gene that suggest this gene being
the human salt receptor is as follows:
[0261] (1) MCOLN3 is expressed in the top of the taste buds, in a
subset of taste sensory cells that do not express TRPM5 (that is,
they are not sweet, bitter or umami) and towards the taste
pore.
[0262] (2) MCOLN3 is also expressed in sensory cells of other
organs, like the ear. It is therefore a `professional` sensory
gene.
[0263] (3) MCOLN3 is strongly expressed in the adrenal glands.
These glands play a very important role in the regulation of sodium
metabolism in the body. MCOLN3 is therefore likely to be a key
molecule in the regulation of sodium metabolism and may regulate
the production of aldosterone by the adrenal glands.
[0264] (4) A human autoimmune disease (Addison's) is characterized
by the destruction of the adrenal glands. One of the telltale
symptoms of this disease is salt craving. The latter is likely to
result from the presence of autoantibodies against MCOLN3, or a
mutation in this gene that disrupts the function of MCOLN3 in taste
buds.
[0265] (5) MCOLN3 is highly expressed by the pituitary glands that
are involved in vasopressin release that regulates urine
production. This further supports the importance of this ion
channel in sodium metabolism and excretion by the body.
[0266] (6) MCOLN3 conducts sodium in electrophysiology studies and
exhibits the right biochemical characteristics predicted for a salt
receptor (detection of K+, Li+ and amiloride sensitivity).
[0267] (7) Neurophysiological experiments (nerve recordings) using
sodium in the varitint mouse (having TRPML3 mutation) indicate that
the Varitint mouse is deficient in its response to sodium (does not
exhibit a robust salty taste response).
[0268] (8) Cell based assays using mammalian cells and amphibian
oocytes which express mutated TRPML3 polypeptides (mutation results
in the ion channel being fixed in the "open" orientation) have
identified TRPML3 enhancers and blockers which should enhance or
block salty taste in taste tests.
[0269] The discovery that MCOLN3 is a human salty taste receptor
has enabled the design of screening assays using cells transfected
with this gene for the purpose of identifying agonists, antagonists
or enhancers (modulators) of the function of this molecule.
[0270] This gene was originally identified by the inventors using a
whole genome screening strategy aimed at identifying genes
specifically expressed in taste cells and screening of a subset
thereof enriched in the top of the human taste buds. The inventors
had deduced from their previous experiments that the top of the
taste buds contain cells that over-express the known taste receptor
genes. In contrast, the bottom of the taste buds contains precursor
cells of the sensory taste bud cells that reside in the top portion
of the taste bud. This database allowed the inventors to identify
many genes specifically expressed by the top (sensory) cells of the
taste buds. One of these genes, MCOLN3, was previously described to
be responsible for the phenotype of a mouse mutant called
varitint-waddler that exhibits early-onset hearing loss, vestibular
defects, pigment abnormalities and perinatal lethality (DiPalma et
al., Mutations in MCOLN3 associated with deafness and pigmentation
defects in varitint-waddler (Va) mice. Proc. Natl. Acad. Sci. USA
99: 14994-14999; 2002). MCOLN3 is expressed in the hair cells and
plasma membrane of stereocilia (in the ears)). This mutation
results in an ala 419 to pro substitution in the fifth
transmembrane domain. The result is a hyperactive MCOLN3 that
results in the death of cells expressing this molecule, like the
hair cells of the ear (hence the deafness of the Va mouse) (Grimm C
et al., Proc Natl. Acad. Sci. USA 104: 19583-8; 2007). Therefore
the inventors anticipated that this mouse would also exhibit salty
taste abnormalities (due to the abnormal MCOLN3 molecule and its
probable effect in the salty taste bud cells of the tongue).
Indeed, as shown in neurophysiology experiments and data contained
herein, these mice when stimulated with salty taste stimuli at
concentrations that should normally elicit salty taste perception
do not respond robustly to salty taste stimuli (nerve recoding
results in Varitint mice discussed infra substantiate that the
TRPML3 mutation, which disrupts the activity of TRPML3 and causes
deafness and balance problems because of the loss of hairy cells in
the inner ear also disrupts salty taste, further substantiating
that a normally functioning TRPML3 or MCOLN3 ion channel is a
prerequisite to salty taste perception).
[0271] Also, since public databases, indicate that MCOLN3 is
expressed strongly in the adrenal and pituitary glands, and since
the adrenal glands represent one of the main sodium metabolism
regulators of the body, TRPML3 likely is significantly involved in
sodium metabolism. (The adrenal glands monitor salt levels of the
blood, and secrete aldosterone (a mineralocorticoid) that regulates
blood pressure and water and salt balance in the body by helping
the kidney retain sodium and excrete potassium. Moreover, when
aldosterone production falls too low, the kidneys are not able to
regulate salt and water balance, causing blood volume and blood
pressure to drop.)
[0272] Therefore, it is also predicted that MCOLN3 is a key
salt/sodium monitoring molecule in the adrenal glands that controls
the production of aldosterone. By contrast, in the tongue, MCOLN3
is expressed by s subset of taste sensory cells located in the top
of the taste buds and is responsible for detecting salty taste.
Either way, MCOLN3 has a pivotal role in detecting salt in various
tissues. This invention therefore constitutes a significant
discovery with significant applications.
[0273] In addition, since TRPML3 or MCOLN3 is also substantially
expressed in the pituitary, the inventors also predict that MCOLN3
is involved in the regulation of vasopressin release. Vasopressin
is a key regulator of urine production through its effects on the
kidneys. Importantly, vasopressin release from the posterior
pituitary is known to be regulated by NaCl concentration. In yet an
additional manner, MCOLN3 appears to be a key regulator of NaCl
metabolism in the body, through its effects on fluid retention,
NaCl sensing and concentration, and blood pressure.
[0274] Therefore this invention identifies and provides functional
(electrophysiological) and immunohistochemistry data and animal
data (neurophysiological studies) which indicate that TRPML3
(MCOLN3) encodes a polypeptide that functions as a primate (e.g.,
human) salty taste receptor and plays a significant role in sodium
sensing and metabolism systemically.
[0275] Based thereon, the present invention has as a significant
focus the development of reliable and efficient assays for
identifying compounds that modulate TRPML3 polypeptides (block,
enhance, activate) as these compounds should modulate salty taste
as well as modulating biological functions relating to TRPML3 such
as sodium transport, absorption and excretion by cells, tissues and
organs as well as having an effect (agonistic or antagonistic) on
aldosterone or vasopressin related activities and conditions
wherein modulation of aldosterone or vasopressin release or
production is therapeutically warranted. As is well known in the
art compounds which agonize or antagonize vasopressin and
aldosterone find well known application in therapy, especially
urinary and cardiovascular conditions as well as conditions
involving edema or aberrant circulation. For example, these
compounds are used in treating hypertension, edema, congestive
heart failure, diabetes and symptoms thereof, among numerous other
conditions.
[0276] In addition, as TRPML3 has an effect on melanocytes and
hairy cells compounds which modulate TRPML3 may be useful in
promoting the proliferation and differentiation of melanocytes, may
be useful in treating pigmentation disorders, may prevent or
restore grey hair or skin to its normal coloration (lost e.g.,
because of disease, age, hormonal dysfunction, UV radiation, or
chemotherapy) and may promote hair follicle growth and
proliferation. Also, TRPML3 modulators may be useful in treating
melanoma as they potentially may selectively kill melanoma cells
expressing TRPML3.
[0277] Based on the foregoing the present invention provides assay
systems that comprise test cells, preferably mammalian cell-based
and oocyte cells, that express a functional TRPML3 which may
comprise a wild-type TRPML3 of any desired species, a mutated
TRPML3 wherein the mutations naturally occur or are introduced by
design, e.g., in order to modify TRPML3 function (enhance or
inhibit) or to maintain it in a fixed open pore orientation to
facilitate its use in modulator screening assays, or it may
comprise a chimeric TRPML3 ion channel or a functional fragment
wherein a domain or extracellular look of TRPML3 is exchanged with
that of another TRPML3 ion channel or another ion channel such as
TRPML1, TRPML2, NALCN or NKAIN3 or another TRP ion channel.
Exemplary mutations to TRPML3 are disclosed herein and may be
designed by a skilled artisan using the information disclosed
herein and methods well known to those skilled in the art. Also, it
may be advantageous, as described herein, to provide TRPML3
encoding nucleic acid sequences which are comprised of host
preferred codons, i.e., codons preferred in the cell wherein the
assays are to be conducted. For example, the inventors provide
infra a TRPML3 gene constituted of human preferred codons in order
to enhance TRPML3 ion channel activity in human (e.g., HEK-293
cells) used in preferred assays according to the invention.
[0278] Preferably, the invention provides mammalian cell-based and
oocyte cell-based assay, preferably high or medium throughput, for
the profiling and screening of the salty taste receptor (TRPML3).
More specifically, the invention provides amphibian oocytes, that
express TRPML3 that can be used in cell-based assays for the
screening of TRPML3 modulators. Also the invention provides
amphibian oocytes that express a functional TRPML3 for use in
functionally characterizing TRPML3 activity, and that may be used
to identify compounds that either enhance or block salty taste
perception (herein referred to as salty taste modulators). These
compounds can be used as ingredients in foods, medicinals and
beverages to enhance, modulate, inhibit or block salty taste. Also,
these compounds have potential therapeutic application, e.g., in
regulating blood pressure, cardiac function, renal function
especially urine production and excretion, in treating Addison's
disease, type IV mucolipidosis, and physiological effects of
aldosterone and/or vasopressin and diseases wherein the
administration of an aldosterone or vasopressin agonist or
antagonist is therapeutically warranted.
[0279] Therefore this invention identifies and provides functional
(electrophysiological), molecular, and immunohistochemistry data
which indicate that TRPML3 (MCOLN3) encodes a polypeptide that
functions as a primate (e.g., human) salty taste receptor.
[0280] Further the present invention provides the use of these
taste specific genes as markers which can be used to enrich,
identify or isolate salt receptor expressing cells.
[0281] Also this invention provides in vitro and in vivo assays
which use TRPML3 (MCOLN3) and TRPML3 expressing cells or TRPML3
transgenic animal models to identify agonist, antagonist or
enhancer compounds which elicit or modulate (block or enhance)
salty taste in primates including humans. These assays use cells
which express TRPML3 alone or cells which express the TRPML3 ion
channel in association with other taste specific polypeptides such
as NALCN or NKAIN3.
[0282] In addition this invention provides transgenic animals,
preferably rodents, and the use thereof to confirm the role of
TRPML3 in salty taste in mammals and in other physiological
functions involving sodium and other ions such as sodium
metabolism, blood pressure, fluid retention and excretion, urinary
function and cardiac function.
[0283] Also this invention provides in vitro and in vivo assays
which use TRPML3 and TRPML3 expressing cells or transgenic animals
in assays, preferably electrophysiological assays in order to
identify therapeutic compounds which alleviate diseases and
conditions involving deficiencies in the expression of this
polypeptide including hyper expression, hypo expression, and
mutations in the TRPML3 polypeptide that affect its ability to
function as a taste specific sodium channel in mammals including
e.g., human and non-human primates. These conditions include by way
of example Addison's disease and other diseases involving or
affected by aberrant aldosterone production or vasopressin release
such as hypertension, hypotension, fluid retention, and impaired
urinary or cardiac function such as arrhythmia, heart attack and
stroke.
[0284] The subject gene was initially identified by the use of the
following methodologies, to identify novel taste-specific
genes:
[0285] 1) Laser capture microdissection (LCM) and RNA
amplification: In laser capture microdissection, a fine laser beam
is used to dissect and purify taste cells from histological
sections. This method isolates taste cells, devoid of contaminating
lingual epithelial cells and connective tissue, and allows one to
perform molecular biology experiments on a highly enriched taste
cell population. In parallel, lingual epithelial cells are isolated
by LCM and used as a negative control devoid of taste cells. LCM is
advantageous to manual or enzymatic dissection of taste papilla
because these crude techniques yield a heterogeneous mixture of
taste and lingual cells in which taste cells comprise 1-20% of
collected material. RNA amplification amplifies total RNAs from
taste cells and lingual cells isolated by LCM up to 1 million-fold
in a non-biased fashion to generate sufficient genetic material to
perform molecular biology studies (gene chips or PCR). We have
found that 5,000 taste cells are sufficient for gene chip
experiments with macaque taste tissue and greater than 10,000 taste
cells are sufficient for PCR experiments with macaque taste
tissue.
[0286] 2) Gene Chips: Gene chips contain most all annotated genes
on a small chip. Hybridizing RNA, isolated and amplified from taste
and lingual cells, to gene chips can be used to determine which
specific genes are expressed in taste cells and not lingual cells
and which specific genes are expressed at higher levels in taste
cells compared to lingual cells. Gene chips experiments were
conducted using paired macaque fungiform (FG) and circumvallate
(CV) taste and lingual samples using Affymetrix rhesus macaque
genome arrays and analyzed using GeneSpring GX v7.3 software
(Agilent Technologies). 5000 fungiform and CV taste and lingual
cells were separately isolated by LCM and total RNA was purified
for each sample. RNA was then amplified and hybridized to gene
chips. Data analyses are performed using two separate algorithms:
Affymetrix Microarray Suite 5 (MAS5) which takes into account both
perfect match and mismatch probes on gene chips, and robust
multi-chip algorithm (RMA) which only takes into account perfect
match probes on gene chips. Taste-specific genes encoding
transmembrane proteins are identified in this analysis.
[0287] 3) PCR: High-throughput PCR is performed in 96 well plates
using primers specific for ion channels in the human/macaque genome
and amplified RNA from human/macaque taste and lingual cells
isolated by LCM. Detection of products of the appropriate size in
taste cells but not lingual cells and DNA sequencing of PCR
products (to confirm gene identity) indicates the ion channel of
interest is a taste-specific gene. Prior to high-throughput PCR
using primers against ion channels identified in the macaque
genome, quality-control PCR reactions are first performed on up to
4 known taste-specific genes and 2 housekeeping genes to ensure
that taste and lingual RNAs are of high quality. Four
taste-specific genes which may be examined are the G alpha protein
gustducin (GNAT3), the sweet receptor component T1R2, the ion
channel TRPM5, and the enzyme phospholipase C isoform beta2
(PLC.beta.2); the two housekeeping genes examined are beta-actin
and GAPDH. Specific expression of the taste genes by taste cells
but not lingual cells plus expression of the ubiquitous
housekeeping genes by both taste and lingual cells indicates high
quality RNA material.
[0288] PCR products are analyzed on agarose gels to determine if
bands of the appropriate size are present in taste cells but not
lingual cells. Genes with this expression pattern are putative
taste-specific genes. All taste-specific genes were cloned and
sequenced to confirm the gene identities.
[0289] 4) In Situ Hybridization: Antisense RNA probes specific for
an individual gene(s) (identified by gene chips or PCR) are
hybridized to tissue sections containing taste cells to determine
if the mRNA transcript for the gene of interest is expressed in
taste cells, specifically in sour, sweet, bitter, and/or umami
cells or in a unique cell type that may be involved in salty taste
detection. In double labeling in situ hybridization, two different
RNA probes are generated to label two different genes, specifically
two different taste-specific genes identified by gene chip and/or
PCR approaches. Alternatively, one probe can be generated to label
a single gene to determine if the gene is expressed in taste cells.
For double labeling studies, the first gene is labeled with a FITC
probe that generates one color in a fluorescent microscope while
the second gene is labeled with a digoxygenin (DIG) probe that
generates a different color in a fluorescent microscope.
Superimposition of probe 1 and probe 2 reveals if genes are
expressed in the same or in different cell types. For example, if a
unique ion channel identified by gene chip or PCR approaches
colocalizes to cells expressing TRPM5, that unique ion channel is
expressed in cells responsible for sweet, bitter, and/or umami
taste. By contrast, if a unique ion channel identified by gene chip
or PCR approaches does not colocalize to cells expressing TRPM5,
that unique ion channel is expressed in a different cell type that
may be responsible for salty taste (or another taste modality) and
that unique ion channel may be directly involved in sodium
detection.
[0290] 5) Immunohistochemistry: Antibodies specific for an
individual protein (whose gene was identified by gene chips or PCR)
are applied to tissue sections containing taste cells to determine
if the protein of interest is expressed in taste cells,
specifically in sour, sweet, bitter, and/or umami cells or in a
unique cell type that may be involved in salty taste detection. In
double labeling immunohistochemistry, two different antibody probes
are used to label two different proteins, specifically two
different taste-specific proteins whose genes were identified by
gene chip and/or PCR approaches. Alternatively, one antibody probe
can be used to label a single protein to determine if the protein
is expressed in taste cells. For double labeling studies, the first
protein is labeled with an antibody at a very dilute concentration
that can only be detected with a sensitive detection method termed
tyramide signal amplification (TSA). The second protein is then
labeled with another antibody and detected using a non-TSA method.
The dilute first antibody cannot be detected by the standard
non-TSA method; therefore, two different antibodies from the same
species (e.g. rabbit polyclonal antibodies) will not cross-react
and, therefore, can be used in double labeling experiments.
Superimposition of protein label 1 and protein label 2 reveals if
proteins are expressed in the same or in different cell types. For
example, if a unique ion channel identified by gene chip or PCR
approaches colocalizes to cells expressing TRPM5, that unique ion
channel is expressed in cells responsible for sweet, bitter, and/or
umami taste. By contrast, if a unique ion channel identified by
gene chip or PCR approaches does not colocalize to cells expressing
TRPM5, that unique ion channel is expressed in a different cell
type that may be responsible for salty taste (or another taste
modality) and that unique ion channel may be directly involved in
sodium detection.
[0291] Further, the identification of the subject ion channel gene
as an ion channel potentially involved in salty taste perception
further included the following rationale to select potential salty
taste receptor or ion channel candidates.
[0292] First taste buds are isolated using LCM as described above
from macaque (Macaca fascicularis). Macaque genes are on average
90-95% identical to human genes and the macaque is an excellent
experimental model for study of human biology including taste. Thus
taste genes identified in the macaque will be highly similar to
their human orthologs and carry out similar functions to those seen
in humans. Using LCM a fine laser beam is used to dissect and
purify taste cells from histological sections. This method isolates
taste cells devoid of contaminating lingual epithelial cells and
connective tissue and allows molecular biology experiments to be
effected on a highly enriched taste cell population. In parallel,
lingual epithelial cells are isolated by LCM and used as a negative
control devoid of taste cells. LCM is advantageous to manual or
enzymatic dissection of taste papilla because these crude
techniques tend to yield a heterogeneous mixture of taste and
lingual cells in which taste cells only comprise about 1-20% of the
collected material.
[0293] Secondly, RNA isolated from taste and non-taste cells is
analyzed using gene chips/microarrays. Gene chips contain most all
annotated genes on a small chip. Hybridizing RNA, isolated from
taste and lingual cells, to gene chips can be used to determine
which specific genes are expressed in taste cells and not lingual
cells as well as which specific genes are expressed at higher
levels in taste cells compared to lingual cells. In order to
identify genes for which probe sets are not functional on gene
chips, gene chips were performed on 21 macaque non-taste tissues.
Probe sets for genes not yielding data above background levels
include both probe sets that do not hybridize efficiently to gene
targets as well as probe sets not represented within the panel of
21 macaque tissues. These genes, representing genes not covered by
the gene chip approach, are analyzed separately by PCR and/or
histology to identify genes, specifically genes encoding
transmembrane proteins, which are expressed in taste cells and not
lingual cells as well as genes expressed at higher levels in taste
cells compared to lingual cells isolated by LCM.
[0294] Third, taste-specific genes identified by gene chips and/or
PCR are examined by histology using double labeling approaches.
With in situ hybridization antisense probes specific for individual
genes are hybridized to tissue sections containing taste cells to
determine if the mRNA transcript for the gene of interest is
expressed in taste cells, specifically in sweet bitter, sour and/or
umami taste cells or in a unique cell type that may be involved in
salt or other taste modality, e.g., fat taste detection. Using
immunohistochemistry antibodies specific for an individual protein
(which gene was identified by gene chips) these antibodies are
applied to tissue sections containing taste cells to determine if
the protein of interest is expressed in taste cells, specifically
in sweet, bitter, sour and/or umami cells or in a unique cell type
that may be involved in salt or fat taste detection. Genes
expressed in taste cells expressing TRPM5, a marker for sweet,
bitter, and umami cells, would encode proteins that may modulate
sweet, bitter and/or umami taste. Genes expressed in taste cells
expressing PKD2L1 or PKD1L3, markers for sour cells, would encode
proteins that may modulate sour taste. Genes expressed in taste
cells expressing neither TRPM5 nor PKD2L1 or PKD1L3 would encode
proteins expressed in a unique cell type that may correspond to a
salt or fat cell. Therefore, genes expressed in a unique taste cell
type may correspond to a salty taste receptor or a fat taste
receptor and may modulate salty or fat taste detection.
[0295] Fourth, using similar LCM procedures and gene chip or PCR
expression methods experiments are conducted to identify which set
of genes are specifically expressed in the top half of taste buds
and not in the bottom half or which are enriched in the top half,
i.e., expressed at least 1.2-1.5 fold higher in the cells
comprising the top half of the taste bud relative to cells in the
bottom half. These genes are preferred candidates for human taste
receptors given their orientation on the taste bud).
[0296] Fifth, taste-specific genes expressed in a unique cell type
are analyzed by use of functional assays including
electrophysiology to determine of gene products expressed in
heterologous systems such as HEK293 cells, CHO cells, or Xenopus
oocytes generate sodium-responsive receptors or sodium-conducting
ion channels. A salt receptor target should respond to sodium ions
at concentrations relevant for human taste (between 20-140 mM
sodium).
[0297] Sixth, to ultimately validate the role of a gene as a salt
receptor, genes meeting the criteria set forth above are advanced
into high-throughput screens to identify enhancers and blockers and
these compounds are tested in salty taste tests to determine if
they augment or repress salty taste perception. In parallel, mouse
knockouts are generated lacking the gene of interest (or expressing
a variant form as herein with the Varitint mouse) and physiological
(nerve recordings) and behavioral (2-bottle preference tests and
gustometer tests) experiments are performed to determine if the
animals are deficient in or lack salty taste perception.
[0298] Therefore, the subject TRPML3 gene was identified as
encoding a polypeptide ion channel that is involved in sodium taste
sensing and likely sodium sensing and metabolism more broadly based
on the following criteria: 1) Genes expressed specifically in taste
cells or at higher levels in taste cells than lingual cells in gene
chip and/or PCR experiments (these are defined as taste-specific
genes); 2) Genes expressed in a unique cell type, that does not
correspond to sweet, bitter, sour, and/or umami cells by histology;
3) Gene products that generate sodium responsive receptors or
sodium channels in electrophysiology or functional experiments; and
4) Enhancers or blockers of gene products modulate salty taste
perception and/or mouse knockouts or expressing inactive or variant
forms of the ion channel gene of interest are deficient in or lack
salty taste responsiveness.
[0299] Using such rationale, methods and protocols a large number
of primate genes were initially identified as taste cell specific.
These genes including TRPML3 are contained in earlier provisional
applications incorporated by reference herein. This large set of
genes given the comprehensive and accurate methodologies used to
identify these genes is predicted by the inventors to be
comprehensive of the genes which are specifically expressed in
primate taste cells.
[0300] From this large group of genes a small subset of taste
specific genes which are taste specific, and which are specifically
expressed or are enriched in the top half of taste buds and which
encode sodium ion channels was identified. In particular three ion
channel taste specific genes were identified, i.e. TRPML3, NKAIN3
and NALCN. Of these 3 genes it was confirmed that TRPML3 encodes an
ion channel that is involved and required for salty taste
perception.
[0301] Specifically, the functional (electrophysiological) and
immunohistochemical data contained in the examples infra and the
data obtained in the Varitint mouse indicate that MCOLN3 (TRPML3)
functions as a salty taste receptor in rodents, humans as well as
other primates and most likely other mammals and also likely plays
a role in other physiological functions involving sodium
metabolism, absorption and excretion such as those relating to
aldosterone production and vasopressin release. The criteria
further that supported the testing of the selected ion channel
genes is summarized in Table 1 below.
TABLE-US-00001 TABLE 1 TB vs LE Top vs Bottom Reference reporting
that this gene encodes Gene Name Ratio TB Ratio a sodium channel
NALCN (aka 11.2 7.2 Cell. 2007 Apr 20; 129(2): 371-83 VGCNL1) The
neuronal channel NALCN contributes resting sodium permeability and
is required for normal respiratory rhythm. Lu B, Su Y, Das S, Liu
J, Xia J, Ren D. Describes NALCN as a sodium leak channel,
consistent with a predicted salt receptor. TRPML3 (aka 10.2 1.6 J
Biol Chem. 2007 Oct 25: [Epub ahead of MCOLN3) print]
Gain-of-function mutation in TRPML3 causes the mouse
varitint-waddler phenotype. Kim H J, Li Q, Tjon-Kon-Sang S, So I,
Kiselyov K, Muallem S. First description of TRPML3 as a channel
permeable to sodium, consistent with a salt receptor. NKAIN3 (aka
3.3 1.5 Hum Mol Genet. 2007 Oct 15: 16(20): 3394-410. FAM77D) Epub
2007 Jul 2 A novel family of transmembrane proteins interacting
with {beta} subunits of the Na,K- ATPase. Gorokhova S, Bibert S,
Geering K, Heintz N. Describes a Drosophila homologue of NKAIN3 as
an amiloride-insensitive sodium channel, consistent with a salt
receptor.
[0302] Therefore, based on the foregoing, the subject invention
generally relates to methods for identifying taste genes, including
genes involved in salty taste perception or other taste perception
such as fat taste perception and the use in screening assays for
identifying human salty taste enhancers and other taste modulatory
compounds and for identifying potential therapeutics that modulate
other taste cell related functions and phenotypes including
diseases and conditions not directly related to taste transduction,
i.e., those relating to aberrant sodium transport, metabolism and
excretion and sensing by different tissues.
[0303] The compounds which modulate TRPML3 have potential
application in modulating human salty taste perception. Compounds
identified for example in electrophysiological assays and their
biologically acceptable derivatives are to be tested in human taste
tests using human volunteers to confirm their effect on human salty
taste perception. In addition compounds identified as potential
therapeutics will be evaluated in appropriate in vitro and in vivo
models depending on the nature of the intended application. For
example compounds identified as potential therapeutics for diabetes
may be evaluated in well known diabetic animal models such the NOD
mouse model or BB rat model. Similarly, compounds identified as
potential therapeutics for IBD or Crohn's disease may be tested in
rodent animal models for IBD or Crohn's disease.
[0304] The cell-based assays used to identify taste, e.g., salty
taste modulatory or therapeutic compounds will preferably comprise
high throughput screening platforms to identify compounds that
modulate (enhance) the activity of genes involved in salty taste
perception using cells that express the genes disclosed herein or
combinations thereof. Additionally, these sequences may be modified
to introduce silent mutations or mutations having a functional
effect such as defined mutations that affect ion (sodium) influx.
As noted above, the assays will preferably comprise
electrophysiological assays effected in amphibian oocytes or assays
using mammalian cells that express a an ion channel according to
the invention using fluorescent ion sensitive dyes or membrane
potential dyes, e.g., sodium-sensitive dyes. Preferably, compounds
that modulate such ion channels are identified by screening using
electrophysiological assays effected with oocytes that express an
ion channel identified herein (e.g., patch clamping or two
electrode voltage clamping).
[0305] Still alternatively, compounds that modulate the subject ion
channels putatively involved in salty taste may be detected by ion
flux assays, e.g., radiolabeled-ion flux assays or atomic
absorption spectroscopic coupled ion flux assays. As disclosed
supra, these compounds have potential application in modulating
human salty taste perception or for modulating other biological
processes involving aberrant or normal ion channel function.
[0306] The subject cell-based assays use mutant nucleic acid
sequences which are expressed in desired cells, preferably oocytes
or human cells such as HEK-293 cells, or other human or mammalian
cells conventionally used in screens for identifying ion channel or
GPCR modulatory compounds. These cells may further be engineered to
express other sequences, e.g., other taste GPCRs, i.e., T1Rs or
T2Rs such as is described in other patent applications by the
present Assignee Senomyx as well as appropriate G proteins. The
oocyte system is advantageous as it allows for direct injection of
multiple mRNA species, provides for high protein expression and can
accommodate the deleterious effects inherent in the overexpression
of ion channels. The drawbacks are however that
electrophysiological screening using amphibian oocytes is not as
amenable to high throughput screening of large numbers of compounds
and is not a mammalian system. As noted, the present invention
embraces assays using mammalian cells, preferably high throughput
assays. These high throughput screening assays typically will use
mammalian cells transfected or seeded into wells or culture plates
wherein functional expression in the presence of test compounds is
allowed to proceed and activity is detected using
membrane-potential fluorescent or ion (sodium) fluorescent
dyes.
[0307] These methods of screening are used to identify TRPML3
modulators, e.g., activators, inhibitors, stimulators, enhancers,
etc., of human salty taste or other taste modalities and potential
therapeutics that target other taste cell functions or phenotypes
using the nucleic acids and proteins, sequences provided herein.
Such modulators can affect salty taste or other taste modalities or
taste cell related functions and phenotypes, e.g., by modulating
transcription, translation, mRNA or protein stability; by altering
the interaction of the ion channel with the plasma membrane, or
other molecules; or by affecting ion channel protein activity.
[0308] Compounds are screened, e.g., using high throughput
screening (HTS), to identify those compounds that can bind to
and/or modulate the activity of a taste receptor or taste ion
channel polypeptide or transporter or fragment thereof. In the
present invention, proteins are recombinantly expressed in cells,
e.g., human cells, or frog oocytes and the modulation of activity
is assayed by using any measure of ion channel, receptor or
transporter function, such as measurement of the membrane
potential, or measures of changes in intracellular sodium or
lithium levels. Methods of assaying ion, e.g., cation, channel
function include, for example, patch clamp techniques, two
electrode voltage clamping, measurement of whole cell currents, and
fluorescent imaging techniques that use ion sensitive fluorescent
dyes and ion flux assays, e.g., radiolabeled-ion flux assays or ion
flux assays.
[0309] An enhancer of a gene identified according to the invention
can be used for a number of different purposes. For example, it can
be included as a flavoring agent to modulate the salty taste of
foods, beverages, soups, medicines, and other products for human
consumption. Additionally, the invention provides kits for carrying
out the herein-disclosed assays.
[0310] In fact, as described in the examples such TRPML3 cell-based
assays have already identified TRPML3 blockers and enhancers which
are to be tested in taste tests to establish their effect on salty
taste perception.
[0311] Also the present invention provides the use of TRPML3 as a
marker which can be used to enrich, identify or isolate salt
receptor expressing cells.
[0312] Also this invention provides in vitro and in vivo assays
which use TRPML3 (MCOLN3) and TRPML3 expressing cells or TRPML3
transgenic animal models to identify agonist, antagonist or
enhancer compounds which elicit or modulate (block or enhance)
salty taste in primates including humans. These assays use cells
which express TRPML3 alone or cells which express the TRPML3 ion
channel in association with other taste specific polypeptides such
as NALCN or NKAIN3 or related TRPML members such as TRPML1 or
TRPML2.
[0313] Further this invention provides transgenic animals,
preferably rodents, and the use thereof to confirm the role of
TRPML3 in salty taste in mammals and in other physiological
functions involving sodium and other ions such as sodium
metabolism, blood pressure, fluid retention and excretion, urinary
function and cardiac function.
[0314] In addition, this invention provides in vitro and in vivo
assays which use TRPML3 and TRPML3 expressing cells or transgenic
animals in assays, preferably electrophysiological assays in order
to identify therapeutic compounds which alleviate diseases and
conditions involving deficiencies in the expression of this
polypeptide including hyperexpression, hyporexpression, and
mutations in the TRPML3 polypeptide that affect its ability to
function as a taste specific sodium channel in mammal including
e.g., human and non-human primates. These conditions include by way
of example Addison's disease and diseases involving aberrant
aldosterone production or vasopressin release such as hypertension,
hypotension, fluid retention, and impaired urinary or cardiac
function such as arrhythmia, heart attach and stroke.
DEFINITIONS
[0315] "Putative salty taste receptor or salty taste ion channel
gene" refers to a gene expressed in taste cells that is not
expressed in lingual cells or is expressed substantially less in
lingual cells that moreover preferably is not expressed in taste
cells that express a T1R, T2R, TRPM5, or PKD2L1/PKD1L3 gene.
Preferably these genes are specifically expressed or are enriched
(expressed at least 1.2-1.5 fold higher) in the top versus the
bottom half of the cells which comprise the taste buds. This
includes chemosensory or taste cells, particularly those of macaque
and likely other mammals. As noted and in the preferred aspect of
the invention TRPML3 has been identified as one such salty taste
receptor (sodium ion channel) polypeptide.
[0316] "TRPML3" or "MCOLN3" refers to a gene or a variant thereof
that is involved in salty taste perception in rodents, humans and
non-human primates and likely other mammals and vertebrates
including birds, reptiles and amphibians. This application contains
immediately preceding the claims exemplary sequences for human,
mouse, bovine, murine, zebrafish, chicken, and other mammalian
species TRPML3 genes and polypeptides. Comparison of these
sequences reveals that the polypeptide sequence of TRPML3
polypeptides are very similar in different species, i.e., mouse and
human TRPML3 are 96% sequence identity and 91% sequence similarity.
Therefore, it should be relatively straightforward for one skilled
in the art to identify TRPML3 genes in other mammalian gene or
genomic and polypeptide libraries. Also, it is likely that other
vertebrates express TRPML3 given the essential role of sodium
metabolism to cell vitality and the general well being of
organisms. According this invention is intended to broadly
encompass salty receptors containing TRPML3 genes and functional
variants such as chimeras of different species, but most preferably
humans and other mammals. Also, the TRPML3 genes and polypeptides
herein specifically embrace TRPML3 mutated genes and fragments
which have been mutated at one or more sites, e.g., in order to
modify (enhance or decrease TRPML3 activity), render the ion
channel more suitable for use in assays such as by modifying the
polypeptide so that the ion channel is fixed in the "open" or
"closed" position or by creating fragments or chimeras wherein a
domain or extracellular loop or a portion thereof of one TRPML3
polypeptide is swapped with that of another ion channel. e.g., a
TRPML3 of a different species or a non-TRPML3 ion channel such as
e.g., TRPML1, TRPML2, NKAIN3, or NALCN. Also, the term TRPML3
polypeptides and nucleic acid sequences specifically encompasses
TRPML3 ion channel polypeptides which posses at least 80% sequence
identity to those disclosed herein, more preferably at least 90%
sequence identity or still more preferably at least 95, 96, 97, 98
or 99% sequence identity to a native TRPML3 polypeptide, e.g., a
native human, non-human primate, rodent (rat, mouse, et al.), dog,
cat, horse, bovine, sheep, etc., TRPML3 polypeptide. TRPML3 nucleic
acid sequences further include all nucleic acid sequences encoding
therefore such as cDNAs, genomic sequences, cRNAs, mRNAs, as well
as single stranded, double stranded and triple stranded nucleic
sequences and their complements. In humans there are 3 major forms
of TRPML3 mRNA in taste buds, pituitary and the adrenal the
sequences of which are contained in the List of sequences before
the claims.
[0317] Also, TRPML3 sequences include sequences that specifically
hybridize to the subject TRPML3 encoding nucleic acid sequences or
their complements which encode a sodium ion channel involved in
salty taste perception and/or sodium transport, metabolism or
excretion. Exemplary hybridization conditions suitable for
identifying other TRPML3 orthologs and related genes are known in
the art and are defined in this application below.
[0318] "Taste Cell" refers to a cell that when mature expresses at
least one receptor, transporter, or ion channel that directly or
indirectly regulates or modulates a specific taste modality such as
sweet, sour, umami, salty, bitter, fatty, metallic or other taste
perception or general taste perception such as taste intensity or
the duration of a taste response. This includes in particular genes
that are expressed specifically in chemosensory or taste cells,
particularly macaque and likely other mammalian taste cells. Taste
cells express mRNA and/or a protein for the gene C6orf15
(chromosome reading frame 15)--also known as STG. This gene has
been described as a taste-specific gene (M. Neira et al. Mammalian
Genome 12: 60-66, 2001) and is among the macaque taste specific
genes reported herein. In addition a mature taste receptor cell
typically will express mRNA and/or protein for alpha ENaC. We have
data (not shown herein) that reveals that alpha ENaC is expressed
in at least sweet, bitter, umami, sour and most likely salty taste
cells. Further, a mature taste receptor cell will typically express
mRNA and/or protein for cytokeratin 19. This protein is only
expressed in mature taste cells and is not found in basal or stem
cells. (L. Wong et al. Chemical Senses 19(3): 251-264, 1994).
Furthermore, taste cells can be identified by those skilled in the
art base on their characteristic morphology. In particular mature
taste receptor taste cells are elongated and spindle-shaped. Also,
a mature taste receptor cell has the apex of the cell (apical
membrane) penetrating into the taste pore thereby gaining access or
exposure to saliva. By contrast, an immature taste cell, e.g., a
basal cell or stem cell is rounded and is not exposed to the taste
pore and saliva. Also, unlike mature taste cells, basal and stem
cells tend to be localized towards the base of taste buds.
[0319] "Chemosensory cells" are cells that are involved in sensing
of chemical stimulants such as tastants and other chemical sensory
stimuli such as odorants. Chemosensory cells herein include in
particular taste receptor cells and cells comprised in the
digestive or urinary tract or other organs that when mature express
one or more taste receptors. For example, gastrointestinal
chemosensory cells are known which express T1Rs or T2Rs and which
cells are likely involved in food sensing, metabolism, digestion,
diabetes, food absorption, gastric motility, et al. In addition,
cells found in the urinary tract likely express salty taste
receptors and are involved in sodium transport, excretion and
functions associated therewith such as blood pressure and fluid
retention. Further, in the digestive system chemosensory cells that
express taste receptors may also express chromogranin A, which is a
marker of secretory granules. (C. Sternini, "Taste Receptors in the
Gastrointestinal Tract. IV. Functional Implications of Bitter Taste
Receptors in Gastrointestinal Chemosensing". American Journal of
Physiology, Gastrointestinal and Liver Physiology.", 292:G457-G461,
2007).
[0320] "Taste-cell associated gene" herein refers to a gene
expressed by a taste cell that is not expressed by lingual cell
that is involved in a taste or non-taste related taste cell
function or phenotype. This includes in particular genes reported
herein and in earlier provisional applications cited herein that
are expressed specifically in chemosensory or taste cells,
particularly those from macaque. Taste cells include cells in the
oral cavity that express taste receptors such as the tongue and
taste cells in other areas of the body that express taste receptors
such as the digestive system and urinary tract. Such genes include
those contained in the tables in the applications incorporated by
reference herein. These genes include genes involved in taste and
non-taste related functions such a taste cell turnover, diseases
affecting the digestive system or oral cavity, immunoregulation of
the oral cavity and/or digestive system, digestive and metabolic
functions involving taste cells such a diabetes, obesity, blood
pressure, fluid retention et al. In referring to the particular
taste specific genes identified therein these genes include the
nucleic acid sequences corresponding the genes contained therein as
well as orthologs thereof and chimeras and variants including
allelic variants thereof. In particular such variants include
sequences encoding polypeptides that are at least 80% identical,
more preferably at least 90% or 95% identical to the polypeptides
encoded by the genes corresponding to the recited genes or to
orthologs thereof, especially human and non-human primate
orthologs. In addition, the genes include nucleic acid sequences
that hybridize under stringent hybridization conditions to a
nucleic acid sequence corresponding to one of the gene sequences
corresponding to the genes in the earlier provisional patent
applications.
[0321] "Cation channels" are a diverse group of proteins that
regulate the flow of cations across cellular membranes. The ability
of a specific cation channel to transport particular cations
typically varies with the valency of the cations, as well as the
specificity of the given channel for a particular cation.
[0322] "Homomeric channel" refers to a cation channel composed of
identical alpha subunits, whereas "heteromeric channel" refers to a
cation channel composed of two or more different types of alpha
subunits. Both homomeric and heteromeric channels can include
auxiliary beta subunits.
[0323] A "beta subunit" is a polypeptide monomer that is an
auxiliary subunit of a cation channel composed of alpha subunits;
however, beta subunits alone cannot form a channel (see, e.g., U.S.
Pat. No. 5,776,734). Beta subunits are known, for example, to
increase the number of channels by helping the alpha subunits reach
the cell surface, change activation kinetics, and change the
sensitivity of natural ligands binding to the channels. Beta
subunits can be outside of the pore region and associated with
alpha subunits comprising the pore region. They can also contribute
to the external mouth of the pore region.
[0324] The term "authentic" or wild-type" or "native" nucleic acid
sequences refer to the wild-type nucleic acid sequences contained
in the Sequence Listing herein as well as splice, allelic and other
variants and other nucleic acid sequences generally known in the
art.
[0325] The term "authentic" or "wild-type" or "native" polypeptides
refers to the polypeptide encoded by the genes and nucleic acid
sequence disclosed in this and earlier provisional patent
applications which are incorporated by reference.
[0326] The term "modified enhance receptor nuclear acid sequence"
or "optimized nucleic acid sequence" refers to a nucleic acid
sequence which contains one or more mutations, particularly those
that affect (inhibit or enhance) gene activity in recombinant host
cells, and most especially oocytes or human cells such as HEK-293
cells. Particularly, these mutations include those that affect
gating by the resultant ion channel containing the mutated subunit
sequence. The ion channel may comprise such mutations in one or
several of the three subunits that constitute the particular ion
channel. The modified nucleic acid sequence for example may contain
substitution mutations in one subunit that affect (impair) gating
function or defective surface expression. The invention embraces
the use of other mutated gene sequences, i.e., splice variants,
those containing deletions or additions, chimeras of the subject
sequences and the like. Further, the invention may use sequences
which may be modified to introduce host cell preferred codons,
particularly amphibian or human host cell preferred codons.
[0327] The term receptor or ion channel protein or fragment
thereof, or a nucleic acid encoding a particular taste receptor or
ion channel or transporter or a fragment thereof according to the
invention refers to nucleic acids and polypeptide polymorphic
variants, alleles, mutants, and interspecies homologs that: (1)
have an amino acid sequence that has greater than about 60% amino
acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid
sequence identity, preferably over a region of at least about 25,
50, 100, 200, 500, 1000, or more amino acids, to an amino acid
sequence encoded by the wild-type nucleic acid or amino acid
sequence of the taste protein, e.g., proteins encoded by the gene
nucleic acid sequences contained in the Table 1 herein as well as
fragments thereof, and conservatively modified variants thereof;
(3) polypeptides encoded by nucleic acid sequences which
specifically hybridize under stringent hybridization conditions to
an anti-sense strand corresponding to a nucleic acid sequence
encoding a gene encoded by one of said genes, and conservatively
modified variants thereof; (4) have a nucleic acid sequence that
has greater than about 60% sequence identity, 65%, 70%, 75%, 80%,
85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,
or higher nucleotide sequence identity, preferably over a region of
at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to
a nucleic acid, e.g., those disclosed herein.
[0328] A putative salty or other taste specific gene or
polynucleotide or polypeptide sequence is typically from a mammal
including, but not limited to, primate, e.g., human; rodent, e.g.,
rat, mouse, hamster; cow, pig, horse, sheep, or any other mammal.
However, as noted the TRPML3 ion channel is expressed in other
(non-mammal) vertebrates where it likely has a similar function.
The nucleic acids and proteins of the invention include either
naturally occurring or recombinant molecules. Typically these genes
will encode proteins that have ion channel activity, i.e., they are
permeable to sodium or lithium. In particular this includes primate
TRPML3 genes and their human and other mammalian orthologs as well
as fragments and variants that retain TRPML3 functionality, i.e.,
behave analogously in electrophysiological assays that monitor
sodium, lithium conductance and responsiveness (lack) to amiloride
as well as in other suitable functional assays.
[0329] By "determining the functional effect" or "determining the
effect on the cell" is meant assaying the effect of a compound that
increases or decreases a parameter that is indirectly or directly
under the influence of a taste gene, preferably salty taste gene
identified herein e.g., functional, physical, phenotypic, and
chemical effects. Such functional effects include, but are not
limited to, changes in ion flux, membrane potential, current
amplitude, and voltage gating, a as well as other biological
effects such as changes in gene expression of any marker genes, and
the like. The ion flux can include any ion that passes through the
channel, e.g., sodium or lithium, and analogs thereof such as
radioisotopes. Such functional effects can be measured by any means
known to those skilled in the art, e.g., patch clamping, using
voltage-sensitive dyes, or by measuring changes in parameters such
as spectroscopic characteristics (e.g., fluorescence, absorbance,
refractive index), hydrodynamic (e.g., shape), chromatographic, or
solubility properties. Suitable electrophysiological assays using
TRPML3 expressing cells are exemplified in the experimental
examples infra.
[0330] "Inhibitors," "activators," and "modulators" of the subject
taste cell expressed polynucleotide and polypeptide sequences are
used to refer to activating, inhibitory, or modulating molecules
identified using in vitro and in vivo assays of these
polynucleotide and polypeptide sequences. Inhibitors are compounds
that, e.g., bind to, partially or totally block activity, decrease,
prevent, delay activation, inactivate, desensitize, or down
regulate the activity or expression of these taste specific
proteins, e.g., antagonists. "Activators" are compounds that
increase, open, activate, facilitate, enhance activation,
sensitize, agonize, or up regulate protein activity. Inhibitors,
activators, or modulators also include genetically modified
versions of the subject taste cell specific proteins, e.g.,
versions with altered activity, as well as naturally occurring and
synthetic ligands, antagonists, agonists, peptides, cyclic
peptides, nucleic acids, antibodies, antisense molecules, siRNA,
ribozymes, small organic molecules and the like. Such assays for
inhibitors and activators include, e.g., expressing the subject
taste cell specific protein in vitro, in cells, cell extracts, or
cell membranes, applying putative modulator compounds, and then
determining the functional effects on activity, as described
above.
[0331] Samples or assays comprising the proteins encoded by genes
identified herein that are treated with a potential activator,
inhibitor, or modulator are compared to control samples without the
inhibitor, activator, or modulator to examine the extent of
activation or migration modulation. Control samples (untreated with
inhibitors) are assigned a relative protein activity value of 100%.
Inhibition of an ion channel is achieved when the activity value
relative to the control is about 80%, preferably 50%, more
preferably 25-0%. Activation of an ion channel is achieved when the
activity value relative to the control (untreated with activators)
is 110%, more preferably 150%, more preferably 200-500% (i.e., two
to five fold higher relative to the control), more preferably
1000-3000% or higher.
[0332] The term "test compound" or "drug candidate" or "modulator"
or grammatical equivalents as used herein describes any molecule,
either naturally occurring or synthetic compound, preferably a
small molecule, or a protein, oligopeptide (e.g., from about 5 to
about 25 amino acids in length, preferably from about 10 to 20 or
12 to 18 amino acids in length, preferably 12, 15, or 18 amino
acids in length), small organic molecule, polysaccharide, lipid,
fatty acid, polynucleotide, siRNA, oligonucleotide, ribozyme, etc.,
to be tested for the capacity to modulate cold sensation. The test
compound can be in the form of a library of test compounds, such as
a combinatorial or randomized library that provides a sufficient
range of diversity. Test compounds are optionally linked to a
fusion partner, e.g., targeting compounds, rescue compounds,
dimerization compounds, stabilizing compounds, addressable
compounds, and other functional moieties. Conventionally, new
chemical entities with useful properties are generated by
identifying a test compound (called a "lead compound") with some
desirable property or activity, e.g., inhibiting activity, creating
variants of the lead compound, and evaluating the property and
activity of those variant compounds. Often, high throughput
screening (HTS) methods are employed for such an analysis.
[0333] A "small organic molecule" refers to an organic molecule,
either naturally occurring or synthetic, that has a molecular
weight of more than about 50 daltons and less than about 2500
daltons, preferably less than about 2000 daltons, preferably
between about 100 to about 1000 daltons, more preferably between
about 200 to about 500 daltons.
[0334] "Biological sample" include sections of tissues such as
biopsy and autopsy samples, and frozen sections taken for
histologic purposes. Such samples include blood, sputum, tissue,
cultured cells, e.g., primary cultures, explants, and transformed
cells, stool, urine, etc. A biological sample is typically obtained
from a eukaryotic organism, most preferably a mammal such as a
primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g.,
guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
[0335] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region (e.g., a gene or sequence
contained in the Table 1 herein), when compared and aligned for
maximum correspondence over a comparison window or designated
region) as measured using a BLAST or BLAST 2.0 sequence comparison
algorithms with default parameters described below, or by manual
alignment and visual inspection (see, e.g., NCBI web site or the
like). Such sequences are then said to be "substantially
identical." This definition also refers to, or may be applied to,
the compliment of a test sequence. The definition also includes
sequences that have deletions and/or additions, as well as those
that have substitutions. As described below, the preferred
algorithms can account for gaps and the like. Preferably, identity
exists over a region that is at least about 25 amino acids or
nucleotides in length, or more preferably over a region that is
50-100 amino acids or nucleotides in length.
[0336] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0337] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0338] A preferred example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nucl. Acids Res. 25:3389-3402 (1977) and Altschul et al.,
J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0
are used, with the parameters described herein, to determine
percent sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a word length M) of 11, an expectation
(E) of 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a word length
of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix
(see Henikoff & Henikoff, Proc. Natl. Acad. Sci., USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands.
[0339] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form, or complements thereof. The term encompasses
nucleic acids containing known nucleotide analogs or modified
backbone residues or linkages, which are synthetic, naturally
occurring, and non-naturally occurring, which have similar binding
properties as the reference nucleic acid, and which are metabolized
in a manner similar to the reference nucleotides. Examples of such
analogs include, without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
[0340] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The
term nucleic acid is used interchangeably with gene, cDNA, mRNA,
oligonucleotide, and polynucleotide.
[0341] A particular nucleic acid sequence also implicitly
encompasses "splice variants." Similarly, a particular protein
encoded by a nucleic acid implicitly encompasses any protein
encoded by a splice variant of that nucleic acid. "Splice
variants," as the name suggests, are products of alternative
splicing of a gene. After transcription, an initial nucleic acid
transcript may be spliced such that different (alternate) nucleic
acid splice products encode different polypeptides. Mechanisms for
the production of splice variants vary, but include alternate
splicing of exons. Alternate polypeptides derived from the same
nucleic acid by read-through transcription are also encompassed by
this definition. Any products of a splicing reaction, including
recombinant forms of the splice products, are included in this
definition. An example of potassium channel splice variants is
discussed in Leicher, et al., J. Biol. Chem. 273(52):35095-35101
(1998).
[0342] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0343] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0344] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0345] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence with respect to the expression product, but not with
respect to actual probe sequences.
[0346] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0347] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine M; and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0348] Macromolecular structures such as polypeptide structures can
be described in terms of various levels of organization. For a
general discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell (3rd ed., 1994) and Cantor and
Schimmel, Biophysical Chemistry Part I: The Conformation of
Biological Macromolecules (1980). "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains, e.g.,
transmembrane domains, pore domains, and cytoplasmic tail domains.
Domains are portions of a polypeptide that form a compact unit of
the polypeptide and are typically 15 to 350 amino acids long.
Exemplary domains include extracellular domains, transmembrane
domains, and cytoplasmic domains. Typical domains are made up of
sections of lesser organization such as stretches of .beta.-sheet
and .alpha.-helices. "Tertiary structure" refers to the complete
three dimensional structure of a polypeptide monomer. "Quaternary
structure" refers to the three dimensional structure formed by the
noncovalent association of independent tertiary units. Anisotropic
terms are also known as energy terms.
[0349] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical, or other physical means. For example,
useful labels include .sup.32P, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens and proteins which can be made detectable,
e.g., by incorporating a radiolabel into the peptide or used to
detect antibodies specifically reactive with the peptide.
[0350] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all.
[0351] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0352] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as form amide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization. Exemplary stringent
hybridization conditions can be as following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C.
[0353] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., and Current Protocols in Molecular Biology, ed.
Ausubel, et al.
[0354] For PCR, a temperature of about 36.degree. C. is typical for
low stringency amplification, although annealing temperatures may
vary between about 32.degree. C. and 48.degree. C. depending on
primer length. For high stringency PCR amplification, a temperature
of about 62.degree. C. is typical, although high stringency
annealing temperatures can range from about 50.degree. C. to about
65.degree. C., depending on the primer length and specificity.
Typical cycle conditions for both high and low stringency
amplifications include a denaturation phase of 90.degree.
C.-95.degree. C. for 30 sec-2 min., an annealing phase lasting 30
sec.-2 min., and an extension phase of about 72.degree. C. for 1-2
min. Protocols and guidelines for low and high stringency
amplification reactions are provided, e.g., in Innis et al. (1990)
PCR Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y.).
[0355] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody will be most
critical in specificity and affinity of binding.
[0356] The term antibody, as used herein, also includes antibody
fragments either produced by the modification of whole antibodies,
or those synthesized de novo using recombinant DNA methodologies
(e.g., single chain Fv), chimeric, humanized or those identified
using phage display libraries (see, e.g., McCafferty et al., Nature
348:552-554 (1990)) For preparation of antibodies, e.g.,
recombinant, monoclonal, or polyclonal antibodies, many technique
known in the art can be used (see, e.g., Kohler & Milstein,
Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72
(1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in
Immunology (1991); Harlow & Lane, Antibodies, A Laboratory
Manual (1988) and Harlow & Lane, Using Antibodies, A Laboratory
Manual (1999); and Goding, Monoclonal Antibodies: Principles and
Practice (2d ed. 1986)).
[0357] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein,
often in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times the background and more typically more than 10 to 100 times
background. Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies rose to a
protein, polymorphic variants, alleles, orthologs, and
conservatively modified variants, or splice variants, or portions
thereof, can be selected to obtain only those polyclonal antibodies
that are specifically immunoreactive with proteins and not with
other proteins. This selection may be achieved by subtracting out
antibodies that cross-react with other molecules. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies
specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Antibodies, A Laboratory Manual (1988) for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity).
[0358] By "therapeutically effective dose" herein is meant a dose
that produces effects for which it is administered. The exact dose
will depend on the purpose of the treatment, and will be
ascertainable by one skilled in the art using known techniques
(see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3,
1992); Lloyd, The Art, Science and Technology of Pharmaceutical
Compounding (1999); and Pickar, Dosage Calculations (1999)).
[0359] Recombinant Expression of Taste (Salty) Gene Identified
Herein
[0360] To obtain high level expression of a cloned gene, such as
those cDNAs encoding the subject genes, one typically subclones the
gene into an expression vector that contains a strong promoter to
direct transcription, a transcription/translation terminator, and
if for a nucleic acid encoding a protein, a ribosome binding site
for translational initiation. Suitable eukaryotic and prokaryotic
promoters are well known in the art and described, e.g., in
Sambrook et al., and Ausubel et al., supra. For example, bacterial
expression systems for expressing the taste specific protein are
available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et
al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545
(1983). Kits for such expression systems are commercially
available. Eukaryotic expression systems for mammalian cells,
yeast, and insect cells are well known in the art and are also
commercially available. For example, retroviral expression systems
may be used in the present invention. As described infra, the
subject putative salty taste affecting genes are preferably
expressed in human cells such as HEK-293 cells which are widely
used for high throughput screening.
[0361] Selection of the promoter used to direct expression of a
heterologous nucleic acid depends on the particular application.
The promoter is preferably positioned about the same distance from
the heterologous transcription start site as it is from the
transcription start site in its natural setting. As is known in the
art, however, some variation in this distance can be accommodated
without loss of promoter function.
[0362] In addition to the promoter, the expression vector typically
contains a transcription unit or expression cassette that contains
all the additional elements required for the expression of the
nucleic acid in host cells. A typical expression cassette thus
contains a promoter operably linked to the nucleic acid sequence
encoding the identified gene and signals required for efficient
polyadenylation of the transcript, ribosome binding sites, and
translation termination. Additional elements of the cassette may
include enhancers and, if genomic DNA is used as the structural
gene, introns with functional splice donor and acceptor sites.
[0363] In addition to a promoter sequence, the expression cassette
should also contain a transcription termination region downstream
of the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes.
[0364] The particular expression vector used to transport the
genetic information into the cell is not particularly critical. Any
of the conventional vectors used for expression in eukaryotic or
prokaryotic cells may be used. Standard bacterial expression
vectors include plasmids such as pBR322 based plasmids, pSKF,
pET23D, and fusion expression systems such as MBP, GST, and LacZ.
Epitope tags can also be added to recombinant proteins to provide
convenient methods of isolation, e.g., c-myc. Sequence tags may be
included in an expression cassette for nucleic acid rescue. Markers
such as fluorescent proteins, green or red fluorescent protein,
.beta.-gal, CAT, and the like can be included in the vectors as
markers for vector transduction.
[0365] Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 vectors, papilloma virus vectors, retroviral
vectors, and vectors derived from Epstein-Barr virus. Other
exemplary eukaryotic vectors include pMSG, pAV009/A.sup.+,
pMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE, and any other vector
allowing expression of proteins under the direction of the CMV
promoter, SV40 early promoter, SV40 later promoter, metallothionein
promoter, murine mammary tumor virus promoter, Rous sarcoma virus
promoter, polyhedrin promoter, or other promoters shown effective
for expression in eukaryotic cells. A particularly preferred
expression system is the BacMam expression system which uses a
baculovirus based vector to express a polypeptide in mammalian
cells, herein a TRPML3 polypeptide expressed in HEK-293 cells.
[0366] Expression of proteins from eukaryotic vectors can also be
regulated using inducible promoters. With inducible promoters,
expression levels are tied to the concentration of inducing agents,
such as tetracycline or ecdysone, by the incorporation of response
elements for these agents into the promoter. Generally, high level
expression is obtained from inducible promoters only in the
presence of the inducing agent; basal expression levels are
minimal.
[0367] The vectors used in the invention may include a regulatable
promoter, e.g., tet-regulated systems and the RU-486 system (see,
e.g., Gossen & Bujard, Proc. Nat'l Acad. Sci. USA 89:5547
(1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al.,
Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155
(1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)).
These impart small molecule control on the expression of the
candidate target nucleic acids. This beneficial feature can be used
to determine that a desired phenotype is caused by a transfected
cDNA rather than a somatic mutation.
[0368] Some expression systems have markers that provide gene
amplification such as thymidine kinase and dihydrofolate reductase.
Alternatively, high yield expression systems not involving gene
amplification are also suitable, such as using a baculovirus vector
in insect cells, with a gene sequence under the direction of the
polyhedrin promoter or other strong baculovirus promoters.
[0369] The elements that are typically included in expression
vectors also include a replicon that functions in the particular
host cell. In the case of E. coli, the vector may contain a gene
encoding antibiotic resistance to permit selection of bacteria that
harbor recombinant plasmids, and unique restriction sites in
nonessential regions of the plasmid to allow insertion of
eukaryotic sequences. The particular antibiotic resistance gene
chosen is not critical, any of the many resistance genes known in
the art are suitable. The prokaryotic sequences are preferably
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells, if necessary.
[0370] Standard transfection methods may be used to produce
bacterial, mammalian, yeast or insect cell lines that express large
quantities of the desired taste specific protein, which are then
purified using standard techniques (see, e.g., Colley et al., J.
Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification,
in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)).
Transformation of eukaryotic and prokaryotic cells are performed
according to standard techniques (see, e.g., Morrison, J. Bact.
132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in
Enzymology 101:347-362 (Wu et al., eds, 1983). Any of the
well-known procedures for introducing foreign nucleotide sequences
into host cells may be used. These include the use of calcium
phosphate transfection, polybrene, protoplast fusion,
electroporation, biolistics, liposomes, microinjection, plasma
vectors, viral vectors and any of the other well known methods for
introducing cloned genomic DNA, cDNA, synthetic DNA or other
foreign genetic material into a host cell (see, e.g., Sambrook et
al., supra). It is only necessary that the particular genetic
engineering procedure used be capable of successfully introducing
at least one gene into the host cell capable of expressing the
gene.
[0371] After the expression vector is introduced into the cells,
the transfected cells are cultured under conditions favoring
expression of the gene. In some instances, such polypeptides may be
recovered from the culture using standard techniques identified
below.
[0372] Assays for Modulators of Putative Taste Cell Specific Gene
Products
[0373] Modulation of a putative taste cell specific protein, can be
assessed using a variety of in vitro and in vivo assays, including
cell-based models as described above. Such assays can be used to
test for inhibitors and activators of the protein or fragments
thereof, and, consequently, inhibitors and activators thereof. Such
modulators are potentially useful in medications or as flavorings
to modulate salty or other taste modalities or taste in general or
for usage as potential therapeutics for modulating a taste cell
related function or phenotype involving one or several of the
identified taste cell specific genes reported herein.
[0374] Assays using cells expressing the subject taste specific
proteins, either recombinant or naturally occurring, can be
performed using a variety of assays, in vitro, in vivo, and ex
vivo, as described herein. To identify molecules capable of
modulating activity thereof, assays are performed to detect the
effect of various candidate modulators on activity preferably
expressed in a cell.
[0375] The channel activity of ion channel proteins in particular
can be assayed using a variety of assays to measure changes in ion
fluxes including patch clamp techniques, measurement of whole cell
currents, radiolabeled ion flux assays or a flux assay coupled to
atomic absorption spectroscopy, and fluorescence assays using
voltage-sensitive dyes or lithium or sodium sensitive dyes (see,
e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988);
Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991); Hoevinsky et
al., J. Membrane Biol. 137:59-70 (1994)). For example, a nucleic
acid encoding a protein or homolog thereof can be injected into
Xenopus oocytes or transfected into mammalian cells, preferably
human cells such as HEK-293 cells or CHO cells. Channel activity
can then be assessed by measuring changes in membrane polarization,
i.e., changes in membrane potential.
[0376] A preferred means to obtain electrophysiological
measurements is by measuring currents using patch clamp techniques,
e.g., the "cell-attached" mode, the "inside-out" mode, and the
"whole cell" mode (see, e.g., Ackerman et al., New Engl. J. Med.
336:1575-1595, 1997). Whole cell currents can be determined using
standard methodology such as that described by Hamil et al.,
Pflugers. Archiv. 391:185 (1981).
[0377] Channel activity is also conveniently assessed by measuring
changes in intracellular ion levels, i.e., sodium or lithium. Such
methods are exemplified herein. For example, sodium flux can be
measured by assessment of the uptake of radiolabeled sodium or by
using suitable fluorescent dyes. In a typical microfluorimetry
assay, a dye which undergoes a change in fluorescence upon binding
a single sodium ion, is loaded into the cytosol of taste cell
specific ion channel-expressing cells. Upon exposure to an agonist,
an increase in cytosolic sodium is reflected by a change in
fluorescence that occurs when sodium is bound.
[0378] The activity of the subject taste cell specific polypeptides
can in addition to these preferred methods also be assessed using a
variety of other in vitro and in vivo assays to determine
functional, chemical, and physical effects, e.g., measuring the
binding thereof to other molecules, including peptides, small
organic molecules, and lipids; measuring protein and/or RNA levels,
or measuring other aspects of the subject polypeptides, e.g.,
transcription levels, or physiological changes that affects the
taste cell specific protein's activity. When the functional
consequences are determined using intact cells or animals, one can
also measure a variety of effects such as changes in cell growth or
pH changes or changes in intracellular second messengers such as
IP3, cGMP, or CAMP, or components or regulators of the
phospholipase C signaling pathway. Such assays can be used to test
for both activators and inhibitors of KCNB proteins. Modulators
thus identified are useful for, e.g., many diagnostic and
therapeutic applications.
[0379] In Vitro Assays
[0380] Assays to identify compounds with modulating activity on the
subject genes are preferably performed in vitro. The assays herein
preferably use full length protein according to the invention or a
variant thereof. This protein can optionally be fused to a
heterologous protein to form a chimera. In the assays exemplified
herein, cells which express the full-length polypeptide are
preferably used in high throughput assays are used to identify
compounds that modulate gene function. Alternatively, purified
recombinant or naturally occurring protein can be used in the in
vitro methods of the invention. In addition to purified protein or
fragment thereof, the recombinant or naturally occurring taste cell
protein can be part of a cellular lysate or a cell membrane. As
described below, the binding assay can be either solid state or
soluble. Preferably, the protein, fragment thereof or membrane is
bound to a solid support, either covalently or non-covalently.
Often, the in vitro assays of the invention are ligand binding or
ligand affinity assays, either non-competitive or competitive (with
known extracellular ligands such as menthol). These in vitro assays
include measuring changes in spectroscopic (e.g., fluorescence,
absorbance, refractive index), hydrodynamic (e.g., shape),
chromatographic, or solubility properties for the protein.
[0381] Preferably, a high throughput binding assay is performed in
which the protein is contacted with a potential modulator and
incubated for a suitable amount of time. A wide variety of
modulators can be used, as described below, including small organic
molecules, peptides, antibodies, and ligand analogs. A wide variety
of assays can be used to identify modulator binding, including
labeled protein-protein binding assays, electrophoretic mobility
shifts, immunoassays, enzymatic assays such as phosphorylation
assays, and the like. In some cases, the binding of the candidate
modulator is determined through the use of competitive binding
assays, where interference with binding of a known ligand is
measured in the presence of a potential modulator. In such assays
the known ligand is bound first, and then the desired compound
i.e., putative enhancer is added. After the particular protein is
washed, interference with binding, either of the potential
modulator or of the known ligand, is determined. Often, either the
potential modulator or the known ligand is labeled.
[0382] In addition, high throughput functional genomics assays can
also be used to identify modulators of cold sensation by
identifying compounds that disrupt protein interactions between the
taste specific polypeptide and other proteins to which it binds.
Such assays can, e.g., monitor changes in cell surface marker
expression, changes in intracellular calcium, or changes in
membrane currents using either cell lines or primary cells.
Typically, the cells are contacted with a cDNA or a random peptide
library (encoded by nucleic acids). The cDNA library can comprise
sense, antisense, full length, and truncated cDNAs. The peptide
library is encoded by nucleic acids. The effect of the cDNA or
peptide library on the phenotype of the cells is then monitored,
using an assay as described above. The effect of the cDNA or
peptide can be validated and distinguished from somatic mutations,
using, e.g., regulatable expression of the nucleic acid such as
expression from a tetracycline promoter. cDNAs and nucleic acids
encoding peptides can be rescued using techniques known to those of
skill in the art, e.g., using a sequence tag.
[0383] Proteins interacting with the protein encoded by a cDNA
according to the invention can be isolated using a yeast two-hybrid
system, mammalian two hybrid system, or phage display screen, etc.
Targets so identified can be further used as bait in these assays
to identify additional components that may interact with the
particular ion channel, receptor or transporter protein which
members are also targets for drug development (see, e.g., Fields et
al., Nature 340:245 (1989); Vasavada et al., Proc. Nat'l Acad. Sci.
USA 88:10686 (1991); Fearon et al., Proc. Nat'l Acad. Sci. USA
89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien
et al., Proc. Nat'l Acad. Sci. USA 9578 (1991); and U.S. Pat. Nos.
5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463).
[0384] Cell-Based In Vivo Assays
[0385] In preferred embodiments, wild-type and mutant taste cell
specific proteins are expressed in a cell, and functional, e.g.,
physical and chemical or phenotypic, changes are assayed to
identify modulators that modulate function or which restore the
function of mutant genes, e.g., those having impaired gating
function. Cells expressing proteins can also be used in binding
assays. Any suitable functional effect can be measured, as
described herein. For example, changes in membrane potential,
changes in intracellular lithium or sodium levels, and ligand
binding are all suitable assays to identify potential modulators
using a cell based system. Suitable cells for such cell based
assays include both primary cells and recombinant cell lines
engineered to express a protein. The subject taste cell specific
proteins therefore can be naturally occurring or recombinant. Also,
as described above, fragments of these proteins or chimeras with
ion channel activity can be used in cell based assays. For example,
a transmembrane domain of an ion channel according to the invention
can be fused to a cytoplasmic domain of a heterologous protein,
preferably a heterologous ion channel protein. Such a chimeric
protein would have ion channel activity and could be used in cell
based assays of the invention. In another embodiment, a domain of
the taste cell specific protein, such as the extracellular or
cytoplasmic domain, is used in the cell-based assays of the
invention.
[0386] In another embodiment, cellular polypeptide levels of the
particular target taste polypeptide can be determined by measuring
the level of protein or mRNA. The level of protein or proteins
related to ion channel activation are measured using immunoassays
such as western blotting, ELISA and the like with an antibody that
selectively binds to the polypeptide or a fragment thereof. For
measurement of mRNA, amplification, e.g., using PCR, LCR, or
hybridization assays, e.g., northern hybridization, RNAse
protection, dot blotting, are preferred. The level of protein or
mRNA is detected using directly or indirectly labeled detection
agents, e.g., fluorescently or radioactively labeled nucleic acids,
radioactively or enzymatically labeled antibodies, and the like, as
described herein.
[0387] Alternatively, protein expression can be measured using a
reporter gene system. Such a system can be devised using a promoter
of the target gene operably linked to a reporter gene such as
chloramphenicol acetyltransferase, firefly luciferase, bacterial
luciferase, .quadrature.-galactosidase and alkaline phosphatase.
Furthermore, the protein of interest can be used as an indirect
reporter via attachment to a second reporter such as red or green
fluorescent protein (see, e.g., Mistili & Spector, Nature
Biotechnology 15:961-964 (1997)). The reporter construct is
typically transfected into a cell. After treatment with a potential
modulator, the amount of reporter gene transcription, translation,
or activity is measured according to standard techniques known to
those of skill in the art.
[0388] In another embodiment, a functional effect related to signal
transduction can be measured. An activated or inhibited ion channel
will potentially alter the properties of target enzymes, second
messengers, channels, and other effector proteins. The examples
include the activation of phospholipase C and other signaling
systems. Downstream consequences can also be examined such as
generation of diacyl glycerol and IP3 by phospholipase C.
[0389] Assays for ion channel activity include cells that are
loaded with ion or voltage sensitive dyes to report activity, e.g.,
by observing sodium influx or intracellular sodium release. Assays
for determining activity of such receptors can also use known
agonists and antagonists for these receptors as negative or
positive controls to assess activity of tested compounds. In assays
for identifying modulatory compounds (e.g., agonists, antagonists),
changes in the level of ions in the cytoplasm or membrane voltage
will be monitored using an ion sensitive or membrane voltage
fluorescent indicator, respectively. Among the ion-sensitive
indicators and voltage probes that may be employed are those
disclosed in the Molecular Probes 1997 Catalog. Radiolabeled ion
flux assays or a flux assay coupled to atomic absorption
spectroscopy can also be used.
[0390] Isolation of Nucleic Acids Encoding TRPML3 Proteins
[0391] This invention relies, in part, on routine techniques in the
field of recombinant genetics. Basic texts disclosing the general
methods of use in this invention include Sambrook and Russell,
Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler,
Gene Transfer and Expression: A Laboratory Manual (1990); and
Current Protocols in Molecular Biology (Ausubel et al., eds.,
1994)).
[0392] Nucleic acids that encode TRPML3 proteins, polymorphic
variants, orthologs, and alleles can be isolated using TRPML3
nucleic acid probes and oligonucleotides under stringent
hybridization conditions, by screening libraries. Alternatively,
expression libraries can be used to clone TRPML3 protein,
polymorphic variants, orthologs, and alleles by detecting expressed
homologous immunologically with antisera or purified antibodies
made against TRPML3 or portions thereof.
[0393] To make a cDNA library, one should choose a source that is
rich in TRPML3 RNA. The mRNA is then made into cDNA using reverse
transcriptase, ligated into a recombinant vector, and transfected
into a recombinant host for propagation, screening and cloning.
Methods for making and screening cDNA libraries are well known
(see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook
et al., supra; Ausubel et al., supra).
[0394] For a genomic library, the DNA is extracted from the tissue
and either mechanically sheared or enzymatically digested to yield
fragments of about 12-20 kb. The fragments are then separated by
gradient centrifugation from undesired sizes and are constructed in
bacteriophage lambda vectors. These vectors and phage are packaged
in vitro. Recombinant phage are analyzed by plaque hybridization as
described in Benton & Davis, Science 196:180-182 (1977). Colony
hybridization is carried out as generally described in Grunstein et
al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
[0395] Alternatively, TRPML3 cRNA encoding TRPML3 may be generated
from TRPML3 DNA plasmids using T7 RNA polymers to transcribe cRNA
in vitro from DNA linearized with appropriate restriction enzymes
and the resultant cRNA microinjected into suitable cells, e.g.,
oocytes, preferably frog oocytes.
[0396] An alternative method of isolating TRPML3 nucleic acid and
its orthologs, alleles, mutants, polymorphic variants, and
conservatively modified variants combines the use of synthetic
oligonucleotide primers and amplification of an RNA or DNA template
(see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide
to Methods and Applications (Innis et al., eds, 1990)). Methods
such as polymerase chain reaction (PCR) and ligase chain reaction
(LCR) can be used to amplify nucleic acid sequences of TRPML3
directly from mRNA, from cDNA, from genomic libraries or cDNA
libraries. Degenerate oligonucleotides can be designed to amplify
TRPML3 homologs using the sequences provided herein. Restriction
endonuclease sites can be incorporated into the primers. Polymerase
chain reaction or other in vitro amplification methods may also be
useful, for example, to clone nucleic acid sequences that code for
proteins to be expressed, to make nucleic acids to use as probes
for detecting the presence of TRPML3 encoding mRNA in physiological
samples, for nucleic acid sequencing, or for other purposes. Genes
amplified by the PCR reaction can be purified from agarose gels and
cloned into an appropriate vector.
[0397] Gene expression of TRPML3 can also be analyzed by techniques
known in the art, e.g., reverse transcription and amplification
mRNA, isolation of total RNA or poly A.sup.+RNA, northern blotting,
dot blotting, in situ hybridization, RNase protection, high density
polynucleotide array technology, e.g., and the like.
[0398] Nucleic acids encoding TRPML3 proteins can be used with
high-density oligonucleotide array technology (e.g., GeneChip.TM.)
to identify TRPML3 protein, orthologs, alleles, conservatively
modified variants, and polymorphic variants in this invention. In
the case where the homologs being identified are linked to
modulation of T cell activation and migration, they can be used
with GeneChip.TM. as a diagnostic tool in detecting the disease in
a biological sample, see, e.g., Gunthand et al., AIDS Res. Hum.
Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med. 2:753-759
(1996); Matson et al., Anal. Biochem. 224:110-106 (1995); Lockhart
et al., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al.,
Genome Res. 8:435-448 (1998); and Hacia et al., Nucleic Acids Res.
26:3865-3866 (1998).
[0399] As noted, a preferred assay for identification of compounds
that modulate, i.e., enhance, inhibit or block TRPML3 comprises an
electrophysiological assay that monitors changes in electrical
current in cells that express TRPML3 that are contacted with at
least one putative TRPML3 modulator (enhancer or inhibitor). These
assays may use any cell that expresses a functional TRPML3. In the
preferred embodiment, the cells will comprise oocytes, preferably
frog oocytes, mammalian cells, yeast cells or insect cells, or
another expression system that is suitable for expressing a
functional TRPML3 ion channel. Preferably, the expression system
will exhibit robust and rapid TRPML3 sodium channel expression and
desirably will not express any or very few endogenous ion channels,
thereby facilitating the identification of compounds that
specifically modulate TRPML3 sodium channel function. Thereby, an
undesirable background response is minimized or eliminated.
Moreover, robust cells, such as oocytes, are desirable as this
enables the cells to be reused in assays according to the
invention. Oocytes have been reported previously to rapidly and
robustly express other functional ion channels (Pascal, CRC Crit.
Rev. Biotech. 22(4):317-87 (1987); Wagner et al., Cell Physiol.
Biochem. 10:1-12 (2000); Canessa et al., Nature 367:463-467
(1994)).
[0400] A particularly preferred electrophysiological assay is a
moderate throughput assay that measures TRPML3 channel function in
frog oocytes by the two-electrode voltage clamp technique. This
robust, fast expression system provides for the expression of up to
millions of ion channels in an oocyte membrane after only about
18-24 hours. Moreover, because oocytes are relatively large (1 mm
in diameter, relatively large compared to most mammalian cells),
they are easy to handle and work with.
[0401] Based on these advantages, a single oocyte can be used to
obtain multiple and repetitive electrophysiological recording.
Also, an oocyte typically expresses few endogenous channels.
Thereby, oocytes allow for repeated direct measurement of the
effect of target compounds on TRPML3 sodium channel function.
[0402] In a preferred two-electrode voltage clamp assay according
to the invention, frog oocytes that have been microinjected with
TRPML3 cRNAs are transferred to glass scintillation vials and
incubated under appropriate conditions to facilitate TRPML3 protein
expression.
[0403] After TRPML3 sodium ion channel expression is obtained,
typically around 24 hours post-cRNA microinjection, TRPML3 function
is measured according to the two-electrode voltage clamp technique
using an appropriate two-electrode voltage measuring device, e.g.,
OpusXpress 6000A parallel oocyte voltage clamp system (MDS
Analytical Technologies). The two-electrode voltage clamp technique
measures the macroscopic electrical current flowing across the
entire oocyte membrane through the TRPML3 sodium ion channels.
Oocytes are punctured with a voltage-sensing electrode and a
current sensing electrode; the voltage, or potential difference
across the oocyte membrane, is clamped to a particular value using
the voltage-sensing electrode and the current, or the flow of ions
across the oocyte membrane, required to maintain the voltage is
measured using the current-sensing electrode. The OpusXpress system
is one example of a commercially available two-electrode voltage
measuring device which is semi-automated and which comprises a
workstation that permits electrophysiological recordings to be made
from eight oocytes simultaneously. This system also provides for
automated oocyte impalement and delivery of target compounds by a
computer-controlled fluid handler that delivers compound into
96-well compound plates. This system can best be described as a
medium or moderate-throughput system as it allows for the
evaluation of up to 100 compounds per week. Of course more
compounds can be screened by the addition of other voltage
measuring devices, as described.
[0404] In this assay system, TRPML3 enhancers will result in an
increase in current passing through the TRPML3 channels in the
oocyte membrane. This value is calculated by a standard formula.
Such assays also may include appropriate negative controls, e.g.,
known TRPML3 inhibitors. Therefore, this compound functions both as
an internal control to verify that oocytes express functional
TRPML3, and allows for the screening of putative TRPML3 enhancers
after compounds are applied (if the target compound is a TRPML3
enhancer it will result in an increase in current passing through
TRPML3 channels in the oocyte membrane).
[0405] Desirably, a % enhancement factor is calculated for each
enhancer. For example, a 100% enhancer increases TRPML3 activity
100% relative to the basal control value (no compound).
[0406] Negative controls are also desirably performed to confirm
that oocytes which are not injected with TRPML3 cRNAs do not
exhibit the same effects.
[0407] More complex analyses are also desirably performed on
compounds that exhibit robust % enhancement valves e.g.,
current/voltage (I/V) curves, competitive experiments and
dose-response curves to determine the concentration at which the
compound exhibits half-maximal activity (EC50 value). These
experiments will further confirm that the effect of the compound is
TRPML3-specific.
[0408] These assays will provide for the identification of TRPML3
modulators, preferably TRPML3 enhancers, which may be used as
additives for foods, beverages, pharmaceuticals and the like in
order to modulate the salty taste associated therewith. Desirably,
a TRPML3 enhancer will exhibit at least 20% enhancement factor,
more preferably at least 50% and even more preferably at least an
100% enhancement factor.
[0409] The compounds tested as modulators of TRPML3 protein can be
any small organic molecule, or a biological entity, such as a
protein, e.g., an antibody or peptide, a sugar, a nucleic acid,
e.g., an antisense oligonucleotide or a ribozyme, or a lipid.
Alternatively, modulators can be genetically altered versions of a
TRPML3 protein. Typically, test compounds will be small organic
molecules, peptides, lipids, and lipid analogs. Preferably, the
tested compounds are safe for human consumption.
[0410] Essentially any chemical compound can be used as a potential
modulator or ligand in the assays of the invention, although most
often compounds can be dissolved in aqueous or organic (especially
DMSO-based) solutions are used. The assays are designed to screen
large chemical libraries by automating the assay steps and
providing compounds from any convenient source to assays, which are
typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays). It will be appreciated that
there are many suppliers of chemical compounds, including ChemDiv
(San Diego, Calif.), Sigma-Aldrich (St. Louis, Mo.), Fluka
Chemika-Biochemica-Analytika (Buchs Switzerland) and the like.
[0411] In the preferred embodiment, moderate or high throughput
screening methods involve providing a small organic molecule or
peptide library containing a significant number of potential TRPML3
modulators (potential activator or inhibitor compounds). Such
"chemical libraries" are then screened in assays, as described
herein, to identify those library members (particular chemical
species or subclasses) that display a desired characteristic
activity. The compounds thus identified can serve as conventional
"lead compounds" or can themselves be used as potential or actual
products. As noted, the preferred oocyte two-voltage clamp
electrode system (a single device) permits about 60 compounds to be
tested per week.
[0412] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0413] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication No. WO
93/20242), random bio-oligomers (e.g., PCT Publication No. WO
92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),
diversomers such as hydantoins, benzodiazepines and dipeptides
(Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)),
vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.
114:6568 (1992)), nonpeptidal peptidomimetics with glucose
scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218
(1992)), analogous organic syntheses of small compound libraries
(Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates
(Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates
(Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid
libraries (see Ausubel, Berger and Sambrook, all supra), peptide
nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),
antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,
14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries
(see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S.
Pat. No. 5,593,853), small organic molecule libraries (see, e.g.,
benzodiazepines, Baum C&EN, January 18, page 33 (1993);
isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and
metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat.
Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No.
5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the
like).
[0414] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek
Biosciences, Columbia, Md., etc.).
[0415] Compounds are screened, e.g., using high throughput
screening (HTS), to identify those compounds that can bind to
and/or modulate the activity of a taste receptor or taste ion
channel polypeptide or transporter or fragment thereof. In the
present invention, proteins are recombinantly expressed in cells,
e.g., mammalian cells, or frog oocytes and the modulation of
activity is assayed by using any measure of ion channel, receptor
or transporter function, such as measurement of the membrane
potential, or measures of changes in intracellular sodium or
lithium levels. Methods of assaying ion, e.g., cation, channel
function include, for example, patch clamp techniques, two
electrode voltage clamping, measurement of whole cell currents, and
fluorescent imaging techniques that use ion sensitive fluorescent
dyes and ion flux assays, e.g., radiolabeled-ion flux assays or ion
flux assays.
[0416] Food and Beverage Compositions Containing TRPML3 Modulatory
Compound Identified Using Disclosed Assays
[0417] The compounds identified using assays which identify TRPML3
modulatory compounds are potentially useful as ingredients or
flavorants in ingestible compositions, i.e., foods and beverages as
wells as orally administered medicinals. Compounds that modulate or
enhance salty taste perception can be used alone or in combination
as flavorants in foods or beverages. In the preferred application,
the modulator will be incorporated into a food or beverage with a
reduced level of sodium and the salty taste of the resulting
product will be similar to that of the high sodium product.
Examples of such foods and beverages include snack foods such as,
potato chips, crackers, soups, dips, soft drinks, packaged meat
products, pretzels among others.
[0418] The salty taste flavor enhancers or blockers identified
according to the present invention can be blended in various foods
and beverages. Other examples of the foods and beverages include a
wide range of foods and beverages, for example, beverages such as
fruit juice beverage, sports drink, vegetable juice, fermented
lactic drink, carbonated beverage, coffee, cocoa, black tea, oolong
tea, green tea, sake, alcohol, and powdered drink; confectionery
products such as candy, chewing gum, tabletted candy, gummy candy,
soda-pop candy, and chocolate; bakery products such as cookie,
biscuit, and bread; deserts such as yogurt and ice cream; snacks
such as potato chips and cracker; stew, curry, soup, dressing, dip,
noodle soup, bouillon stock, miso, instant bouillon, sauce,
bouillon, jam, sprinkling topping, Japanese pancake, miso soup,
pickles, rice-ball topping, topping for tea and rice, semi-cooked
or cooked foods such as wheat, buckwheat, and Chinese noodles, or
the chilled and frozen foods thereof; instant foods such as instant
noodle; and seasoning such as mixed powdery seasonings and
mayonnaise.
[0419] Among the foods and beverages to which a salt flavor
enhancer is compounded, particularly favorable for improvement in
body are nutritious and nourishing drinks, functional drinks
including nutrition supplement drinks, snack products such as
potato chips and flavored cracker, savory processed foods such as
curries, stews and soups, and the like. The forms of the savory
processed food above include cooked and semi-cooked foods and the
retort-pouch, chilled or frozen foods thereof.
[0420] The amount of the salt flavor enhancer or blocker of the
present invention blended may vary according to the form of the
salt flavor enhancer or blocker and the food or beverage to be
blended with, but is preferred in the range of 0.000001 to 1.0 wt
%, more preferably 0.0001 to 0.1 wt %, and still more preferably
0.00001 to 0.01 wt % with respect to the food or beverage. The salt
flavor enhancer or blocker of the present invention can be blended
by any one of known methods.
[0421] Alternatively, compounds that block or inhibit salty taste
perception can be used as ingredients or flavorants in foods that
naturally contain high salt concentrations in order to block or
camouflage the salty taste thereof. These materials include sports
beverages and other compositions wherein a high amount of
electrolytes including sodium are present, e.g., for medicinal or
replacement purposes after sickness or vigorous exercise which
depletes the electrolyte balance.
[0422] The compositions for ingestion which may include a TRPML3
modulatory compounds according to the invention will include
compositions for ingestion by humans, animals (domesticated, zoo
animals, pets) and will include foods, beverages, medicaments,
neuticeuticals and cosmetics.
[0423] The amount of such TRPML3 modulatory compound(s) will be an
amount that yields the desired degree of salty taste perception. Of
course compounds used in such applications will be determined to be
safe for human consumption and to be acceptable in human taste
tests.
[0424] Preferred Assay Embodiment--Measurement of TRPML3 Currents
in Oocytes Using Two-Electrode Voltage Clamp Electrophysiological
Recordings.
[0425] Electrophysiological Assay for Identifying TRPML3 Modulators
Using Amphibian Oocytes that Express Functional Human TRPML3
[0426] The oocyte expression system has intrinsic advantages
(expression levels, robust, low endogenous ion channel expression)
that render it useful to examine the effects of compounds on sodium
transport through TRPML3 channels. These compounds are candidates
for enhancing salty taste perception. The oocyte expression system
has been used earlier for the rapid and robust expression of ion
channels in functional studies (Dascal, CRC Crit. Rev. Biochem.
(1987) 22(4): 317-387; Wagner, et al, Cellular Physiology and
Biochemistry (2000) 10:1-12; Canessa, et al, Nature (1994) 367:
463-467). Therefore, this system was selected for use in a
two-electrode voltage clamp assay using methods and materials as
described below.
[0427] The oocyte expression system is comprised of the following
steps and methodologies, which collectively comprise the screen for
TRPML3 enhancers: frog surgery and oocyte isolation, cRNA
preparation, oocyte microinjection, and measurement of TRPML3
currents in oocytes using two-electrode voltage clamp
electrophysiological recordings. The following references describe
general practices for frog surgery and oocyte isolation
(Marcus-Sekura, et al, Methods in Enzymology (1987) 152: 284-288;
Goldin, Methods in Enzymology (1992) 207: 266-279), cRNA
preparation (Swanson, et al, Methods in Enzymology (1992) 207:
310-319; Goldin, et al, Methods in Enzymology (1992) 207: 279-297),
oocyte microinjection (Matten, et al, Methods in Enzymology (1995)
254: 458-466; Hitchcock, et al, Methods in Enzymology (1987) 152:
276-284), and two-electrode voltage clamp electrophysiological
recording (Stuhmer, Methods in Enzymology (1992) 207: 319-339;
Wagner, e t a 1, Cellular Physiology and Biochemistry (2000)
10:1-12). Each of these methodologies, as they pertain to the
screen for TRPML3 enhancers, is described in further detail
below.
[0428] Frog Surgery and Oocyte Isolation
[0429] Female Xenopus laevis South African clawed frogs greater
than or equal to 9 cm in length are obtained from NASCO (Fort
Atkinson, Wis.). Frogs are anesthetized in 0.15% MS-222 (tricaine
or ethyl-3-aminobenzoate methanesulfonate; Sigma) in distilled
water and placed on ice. Using sterile surgical tools, sequential
1-2 cm incisions are made in the abdomen through both the outer
skin layer and the inner peritoneal layer. Excised ovarian lobes
(containing 1000-2000 oocytes) are placed in OR-2 calcium-free
media (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl.sub.2, 5 mM HEPES pH 7.5
with NaOH) and sequentially digested with 2 mg/ml collagenase type
IA (Sigma), prepared immediately before use, for 45 min followed by
1 mg/ml collagenase type IA for 15 min on a rocking platform at
room temperature. After enzymatic digestion, at which point the
majority of oocytes are released from the ovarian lobes, oocytes
are rinsed in OR-2 without collagenase and transferred to a Petri
dish containing Barth's saline (88 mM NaCl, 2 mM KCl, 0.82 mM
MgSO.sub.4, 0.33 mM Ca(NO.sub.3).sub.2, 0.41 mM CaCl.sub.2, 2.4 mM
NaHCO.sub.3, and 5 mM HEPES pH 7.5; Specialty Media) supplemented
with 2.5 mM sodium pyruvate. Mature stage V or VI oocytes (.about.1
mm diameter) containing distinct animal poles, corresponding to the
dark side of the egg containing melanin pigment granules, and
vegetal poles, corresponding to the light side of the egg
containing yolk proteins, are selected for microinjection Frogs are
sutured using a C6 needle with a 3-0 black braid suture (Harvard
Apparatus) and reused for oocyte isolation following a 2-3 month
recovery period.
[0430] cRNA Preparation
[0431] TRPML3 cRNA is generated using the mMESSAGE mMACHINE kit
according to the manufacturer's instructions (Ambion) from human
TRPML3 DNA plasmids described in WO 02/087306 A2 using T7 RNA
polymerase to transcribe cRNA in vitro from DNA linearized with
restriction enzymes. cRNA quality is checked by denaturing agarose
gel electrophoresis and spectrophometric absorbance readings at 260
and 280 nm to ensure that full-length, non-degraded cRNA is
generated.
[0432] Microinjection
[0433] Microinjection needles are pulled on a Model P-97
Flaming/Brown Micropipette Puller (Sutter Instrument Co.) using
borosilicate glass capillaries (World Precision Instruments Inc.),
back-filled with mineral oil (Sigma), and then front-filled with
TRPML3 cRNA using a Nanoliter 2000 injector with a Micro4
MicroSyringe Pump Controller (World Precision Instruments). Oocytes
are microinjected in the animal pole with 10-25 nl containing 10-25
ng of human TRPML3 cRNA. Following microinjection, oocytes are
transferred to glass scintillation vials containing Barth's
solution supplemented with 2.5 mM sodium pyruvate and incubated at
18-19.degree. C. overnight under normal atmospheric conditions.
During this time, the oocytes translate injected TRPML3 cRNA into
protein.
[0434] Measurement of TRPML3 Currents in Oocytes Using
Two-Electrode Voltage Clamp
[0435] Twenty-four to forty-eight hours following microinjection of
10-25 ng human TRPML3 cRNA, TRPML3 function is measured in oocytes
using the two-electrode voltage clamp technique on an OpusXpress
6000A parallel oocyte voltage clamp system (MDS Analytical
Technologies). The two-electrode voltage clamp technique is an
electrophysiology method that measures the macroscopic electrical
current flowing across the entire oocyte membrane though protein
channels (Stuhmer, Methods in Enzymology (1992) 207: 319-339).
Oocytes are impaled with a voltage-sensing electrode and a
current-sensing electrode; the voltage, or potential difference
across the oocyte membrane, is clamped to a particular value using
the voltage-sensing electrode and the current, or the flow of ions
across the oocyte membrane, required to maintain that voltage is
measured using the current-sensing electrode. The OpusXpress system
is a semi-automated two-electrode voltage clamp workstation that
allows recordings to be made from 8 oocytes simultaneously. Oocyte
impalement is automated and compound delivery is performed by
computer-controlled fluid handlers from 96-well compound
plates.
[0436] Oocytes are placed in the OpusXpress system and bathed in
ND-96 solution (96 mM NaCl, 2.5 mM KCl, 1 mM CaCl.sub.2, 1 mM
MgCl.sub.2, and 5 mM HEPES pH 7.5 with NaOH). Oocytes are impaled
with voltage-sensing and current-sensing electrodes, pulled on a
Model P-97 Flaming/Brown Micropipette Puller (Sutter Instrument
Co.) using borosilicate glass capillaries (World Precision
Instruments Inc.) and back-filled with 3M KCl, containing silver
chloride wires. Electrodes exhibit resistances between 2-10 Mohm
for voltage-sensing electrodes and between 0.5-2 Mohm for
current-sensing electrodes. Following impalement, oocytes are
voltage clamped to -60 mV and experimental recordings are
initiated. Data are acquired at 50 Hz and low-pass filtered at 5 Hz
using a 4-pole Bessel filter.
[0437] A flowchart illustrating the sequence of experiments
performed to examine the effect of a compound on TRPML3 function is
depicted in FIG. 10, including screening at a holding potential of
-60 mV, I/V curves, NDMG competition tests, dose-response curves,
and testing uninjected oocytes.
[0438] Preferred Assay Embodiment--Measurement of TRPML3 Currents
in Oocytes using Two-Electrode Voltage Clamp Electrophysiological
Recordings and Codon Optimized TRPML3 Sequence
[0439] Codons comprise three nucleotides that encode a specific
amino acid in a protein sequence. Since there are 61 different
codon nucleotide triplets that encode 20 amino acids, most amino
acids can be encoded by more than one codon. Codon optimization,
the use of the favored codon for each amino acid in a particular
species, can improve the functional expression of proteins by
increasing the speed and accuracy of translation without changing
the protein sequence.
[0440] A codon-optimized version of the human TRPML3 gene was
generated that is 76.4% homologous to non-codon optimized TRPML3 at
the DNA level (FIG. 19). Codons were optimized for optimal
translation of human sequences. We also generated an active form of
TRPML3 (A419P TRPML3) by mutating alanine 419 to proline in the
5.sup.th transmembrane domain. This mutation results in TRPML3
channels that are in an open confirmation (Xu et al. PNAS 104(46):
18321-18326, 2007; Grimm et al. PNAS 104(49): 19583-19588, 2007;
Nagata et al. PNAS 105(1): 353-358, 2008; Kim et al. J. Biol. Chem.
282(50): 36138-36142, 2007); therefore, A419P TRPML3 is
particularly useful for identification of TRPML3 blockers.
[0441] Wild-type (non-codon optimized), codon-optimized, and A419P
TRPML3 are expressed in oocytes and sodium currents were measured.
FIG. 20 illustrates that wild-type TRPML3 yields low sodium current
levels, codon-optimized wild-type TRPML3 yields intermediate sodium
current levels, and A419P TRPML3 yields high sodium current levels.
Thus, codon-optimized wild-type TRPML3 and A419P TRPML3 facilitate
screening for compounds that modulate TRPML3 function.
[0442] Codon-optimized wild-type TRPML3 can be used to screen for
compounds that open (enhance) TRPML3 function as described
previously. The data in the examples infra demonstrates how the
electrophysiology oocyte assay using these sequences can be used to
identify TRPML3 enhancers that are candidate human salty taste
enhancers, and how wild-type codon-optimized TRPML3 facilitates
identification of TRPML3 enhancers in the oocyte electrophysiology
assay.
[0443] Preferred Assay Embodiment--TRPML3 Mammalian Cell
Electrophysiological Assays
[0444] The assays may be effected using different mammalian cells.
In a preferred exemplary embodiment immortalized mammalian cells or
tissue culture cells such as human embryonic kidney cells (HEK293
cells) or Chinese hamster ovary cells (CHO cells) are used to
examine the effects of compounds on sodium transport through
exogenously expressed human TRPML3 cation channels. These compounds
are candidates for modulating salty taste perception. The
expression of ion channels in mammalian tissue culture cell lines
is widely used for the rapid and robust expression of ion channels
for functional studies. Advantages of using cultured mammalian
cells as an expression system include: multiple and well
established methods for introducing cDNA of interest into cells
including the ability to generate stable cell lines, relative ease
to perform patch clamp experiments, high level of currents in
comparison to the current stemming from an endogenously expressed
ion channel; the ability to directly measure ion channel
function.
[0445] Electrophysiological recording from mammalian tissue culture
cell lines is comprised of the following steps and methodologies,
familiar to those skilled in the art of electrophysiology, tissue
culture and molecular biology: cell maintenance in culture, cDNA
preparation and purification, introduction of cDNA into cells by
transfection and/or viral transduction, stable clone selection, and
patch-clamp electrophysiology. The following references describe
general practices for patch clamp electrophysiology of mammalian
cells (Sackman B. and E. Neher (eds.). 1995. Single-channel
recording 2.sup.nd Ed.; Hille B. 2001. Ion channels of excitable
membranes, 3.sup.rd Ed.).
[0446] Measurement of TRPML3 Currents in Mammalian Cells Using
Whole Cell Voltage Clamp Electrophysiological Recordings
[0447] The whole cell voltage clamp technique is an
electrophysiology method that measures the macroscopic electrical
current flowing across the entire plasma membrane though protein
channels. Live mammalian cells are placed in a special microscope
chamber containing extracellular solution. Guided by a
micromanipulator and under visual control, a small diameter glass
pipette filled with an electrically conductive salt solution is
first attached to the cell membrane using gentle negative pressure
resulting in a high resistance gigaOhm seal. The membrane patch
within the pipette is disrupted using further suction resulting in
the whole cell patch clamp configuration. The whole cell patch
clamp configuration allows the combined measurement of all ion
channels proteins within the membrane or macroscopic current. Using
the patch clamp amplifier in combination with the whole-cell patch
clamp configuration allows the operator to control the voltage, or
potential difference across the entire cell membrane, as well as
the both the internal and external ionic composition of the cell.
Thus the technique provides a highly sensitive and flexible
platform for the biophysical study of ion channels properties
including (but not limited to) voltage dependence, activation and
deactivation kinetics and permeability to different ions as well as
a screening platform for ion channel blockers, enhancers and
modulators. Utilization of the computer controlled patch clamp
amplifier in concert with a valve controller allows for voltage
protocols to be automatically executed and for the extracellular
solution to be rapidly exchanged. Thus a single cell may be subject
to multiple voltage protocols and compound additions.
[0448] Prior to any electrophysiological assay the efficient
delivery of TRPML3 cDNA into mammalian tissue culture cells must be
obtained. This may be achieved in at least three ways: 1) transient
transfection of TRPML3 cDNA using lipid based methods 2)
transduction using viral infection such as baculovirus, adenovirus,
and lentivirus 3) stable expression of cDNA through the stable
incorporation of TRPML3 into a chromosome and selection of clones
expressing TRPML3. The electrophysiological protocols utilized to
screen mammalian cells for enhancers and blockers of TRPML3
currents are analogous to those previously described for Xenopus
oocytes. In brief these include current voltage analysis, NMDG
competition and dose responses for candidate TRPML3 blockers and
enhancers. In addition, the whole-cell patch clamp
electrophysiological technique can overcome some limitations
imposed by two electrode voltage clamping of oocytes. For example,
the smaller size of mammalian cells allows for more detailed
biophysical analysis of fast processes such as the effects of
compound on activation and deactivation kinetics. Also, the ability
to control the intracellular solution of the cell allows
measurement of any changes in channel permeability due to compound
addition. Finally, the ability for cell attached, inside-out and
outside-out patch configurations allows for the ability to measure
single channel currents allowing detailed characterization of the
mechanism of action of any enhancer or blocker.
[0449] Use of CHO cells for functional expression of Wild Type
TRPML3 and screening of salty taste modulators. Previous reports
show little or no function for wild type (WT) TRPML3 in HEK293
cells using patch clamp assay (Xu et al. PNAS 104(46): 18321-18326,
2007; Grimm et al. PNAS 104(49): 19583-19588, 2007; Nagata et al.
PNAS 105(1): 353-358, 2008; Kim et al. J. Biol. Chem. 282(50):
36138-36142, 2007). In contrast the A419P mutant TRPML3 is believed
to be unregulated in HEK293 cells resulting in robust currents when
transiently expressed. In FIG. 24A, HEK293 cells are transiently
transfected with WT and A419P mutant TRPML3 and their currents
assayed by a series of voltage steps from -100 to +60 mV in order
to generate a current-voltage relation plot (I/V plot). Expression
of A419P mutant TRPML3 channel results in large, inward rectifying
currents compared to WT. We describe in the examples infra the use
of an alternative cell line such as CHO cells that allow for
increased function of WT TRPML3 (FIG. 24 B). In addition, we show
the average macroscopic currents and inward rectification are the
same for WT and A419P TRPML3 channels. Thus, the use of specific
cell lines such as CHO cells allows for more efficient functional
expression of WT TRPML3 in a mammalian system providing a platform
for salty taste modulator screening. In FIG. 25, we demonstrate the
practice of using WT and A419P mutant TRPML3 channels expressed in
CHO cells to test and study potential salty taste enhancers and
blockers. In whole cell electrophysiological assays, an enhancer of
TRPML3 would be observed as an increase in inward current when the
cell is voltage clamped to negative potentials. The I/V analysis in
FIG. 25 A shows WT TRPML3 being enhanced by compound. In the same
assay, a blocker of TRPML3 results in a reduction of inward current
at negative potentials. In FIG. 25 B, the A419P mutant TRPML3
channel expressed constitutively in CHO cells is blocked by the
compound Gadolinium chloride, an inhibitor of many ion
channels.
[0450] Use of Codon Optimized cDNA for Efficient Expression of
TRPML3 in Mammalian Cells and Screening of Salty Taste
Modulators.
[0451] Codons comprise three nucleotides that encode a specific
amino acid in a protein sequence. Since there are 61 different
codon nucleotide triplets that encode 20 amino acids, most amino
acids can be encoded by more than one codon. Codon optimization,
the use of the favored codon for each amino acid in a particular
species, can improve the functional expression of proteins by
increasing the speed and accuracy of translation without changing
the protein sequence. As stated previously a codon-optimized
version of the human TRPML3 gene was generated that is 76.4%
homologous to non-codon optimized TRPML3 at the DNA level (FIG.
19). Codons were optimized for optimal translation of human
sequences. Previously, we showed the WT TRPML3 channel did not
express functional channels efficiently in HEK293 cells (FIG. 24
A). We demonstrate in FIG. 26 that use of the codon optimized
TRPML3 largely overcomes the expression problems observed in HEK293
cells. When codon-optimized WT TRPML3 is expressed in HEK293 cells
by either transient transfection or using baculovirus transduction
(FIG. 26 B), robust currents are observed with similar properties
as the A419P TRPML3 mutant channel. Thus the use of codon optimized
TRPML3 allows for improved functional expression of WT TRPML3 ion
channels in HEK293 cells providing a platform for the screening of
salty taste modulators. In FIG. 26C, we demonstrate the practice of
using codon optimized WT TRPML3 channels to test and study
potential salty taste modulators. In this experiment codon
optimized WT TRPML3 cDNA is delivered to mammalian cells via
transduction by baculovirus. Codon optimized WT TRPML3 mediated
inward currents are enhanced by the TRPML3 activating compound
(FIG. 26C)
[0452] Use of TRPML3 Heteromultimers for Expression and Screening
of Salty Taste Modulators.
[0453] The TRPML3 ion channel subunit is a member of the larger
6TMD ion channel family of ion channel subunits. Similar to other
6TMD ion channels, it is believed that up to 4 TRPML subunits are
necessary to generate a single ion channel (Hille B. 2001. Ion
channels of excitable membranes, 3.sup.rd Ed.; Venkatachalam et al.
J Biol. Chem. 2006 Jun. 23; 281(25):17517-27). Functional channels
may be composed entirely of the same subunit (homomeric) or
associate with closely related subunits (heteromeric). A common
feature of heteromeric channels is that they often posses
intermediate biophysical functions as compared with their homomeric
counterparts, thus increasing the potential functional diversity of
the channel. Activity may also be modulated by different
composition of subunits through changes in plasma membrane
trafficking, and post-translational modification, such as
phosphorylation, ubiquination, and glycosylation. The study of
heteromeric channels in mammalian cells can be achieved by the
delivery of multiple channel subunit cDNAs via co-transfection in
mammalian cells or co-injection of cRNA in Xenopus oocytes. In
addition, multimerization can also be achieved by covalently
linking channel subunit cDNAs together, generating stable cell
lines expressing multiple cDNAs, or viral transduction with
multiple viruses which deliver cDNA for multiple channel
subunits.
[0454] In practice, multimerization of WT and A419P TRPML3 channel
subunits can be used to increase the level of surface activity
(FIG. 27). As already shown, channels consisting exclusively of
A419P TRPML3 subunits express functional channels in HEK293 cells
(FIG. 27 A). In the same cells, when non codon optimized WT TRPML3
subunits are expressed, no currents are observed even when using
3-fold the amount of cDNA (FIG. 27 B). In contrast, coexpression of
A419P TRPML3 cDNA with WT TRPML3 cDNA in HEK293 cells results in a
cooperative effect resulting in larger currents than would be
predicted by simple addition of two separate channel populations
(FIG. 27 C-D). This data suggests that functional channels
consisting of WT and A419P mutant subunits exist at the membrane
and may be utilized for TRPML3 enhancer and blocker assays. As
shown in this example, using limited amounts of A419P cDNA versus
WT should increase the proportion of WT TRPML3 subunits within the
channel tetramer, possibly conferring intermediate biophysical
function to the channel. Therefore since it has been suggested that
homomeric A419P TRPML3 channels may already have a high probability
of opening (Po) (Xu et al. PNAS 104(46): 18321-18326, 2007), use of
heteromeric A419P/WT TRPML3 channels may be more suitable for the
screening of TRPML3 enhancers.
[0455] Preferred Assay Embodiment--Monitoring of Variant TRPML3
Function Using Membrane Potential Dyes.
[0456] Specific cell-based assays for the discovery of TRPML3
modulators were developed which that could ultimately be used in
food and beverages to modulate saltiness perception. A419P-TRPML3
function was monitored in HEK293 cells transiently transfected or
transduced with the gene encoding A419P-TRPML3 and using specific
membrane potential dyes (FMPs; Molecular Devices).
[0457] In one embodiment of the invention, the mammalian or frog
oocyte cell expressing the TRPML3 or a variant, fragment or
functional equivalent is preloaded with a membrane potential
fluorescent dye or a sodium fluorescent dye. The cell is then
contacted with a TRPML3 putative modulator compound in the presence
of sodium or lithium. Cation-mediated changes in fluorescence of
the cell in the presence of the putative modulator are compared to
changes in the absence of the modulator to determine the extent of
TRPML3 modulation.
[0458] Alternatively, the mammalian cell may be transfected with a
functional TRPML3 splice variant and fragments. The cells are then
seeded in the well of a multi-well plate and incubating for a time
sufficient to reach at least about 70% confluence. The cells are
then dye-loaded with a membrane potential dye and contacted with at
least one putative modulating compound and sodium. Any changes in
fluorescence of the membrane potential dye due to modulator/TRPML3
interactions are monitored using a fluorescence plate reader or
voltage intensity plate reader. A putative modulator of salty taste
may then be identified by the changes in fluorescence.
[0459] Preferred Assay Embodiment--IonWorks TRPML3 Patch Clamp
Assay
[0460] The IonWorks automated patch clamp system is used to examine
the effects of compounds on sodium transport through human TRPML3
cation channels. These compounds are candidates for modulating
salty taste perception. The IonWorks system is widely used for
high-throughput electrophysiology. With its 384-well format the
IonWorks system can examine thousands of compounds per day, the
highest throughput of any automated electrophysiology system. The
IonWorks instrument has the advantage that it can be run in the
standard mode where each well corresponds to a single cell or in
the population patch clamp (PPC) mode where each well gives the
average current of 64 cells thus increasing the overall success
rate and reducing well to well variability. Other advantages of the
IonWorks system are that it uses mammalian cells and it provides a
direct measurement of ion channel function by recording ion
currents.
[0461] Measurement of TRPML3 Currents in CHO-K1 Cells Using the
IonWorks Patch Clamp Assay
[0462] Different versions of human TRPML3 can be assayed, included
but not limited to a wild-type version and a gain of function
mutant version encoding an A419P substitution. TRPML3 can be
expressed in CHO-K1 cells by one of three methods: (i) transient
transfection (ii) BacMam transduction or (iii) a stable
transfection and TRPML3 function measured using the perforated
patch clamp technique on an IonWorks Quattro instrument (MDS
Analytical Technologies). Cells are dissociated with Detachin cell
detachment solution (Genlantis), centrifuged, and resuspended in
external recording buffer (150 mM NaCl, 2 mM KCl, 1.5 mM
CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES pH 7.4 with NaOH).
Dissociated cells are added to the 384-well PPC plate where each
well has 64 holes in the substrate with each hole having a diameter
of 1-2 .mu.m. One cell lands on each hole and negative pressure is
used to form a high mega-Ohm seal between the cell and the hole.
After the seal has formed the perforated patch clamp technique is
used to gain electrical access to the inside of the cell. In this
technique, amphotericin, a pore-forming antibiotic, is applied
below the hole and forms small pores on an isolated, exposed
section of the plasma membrane resulting in electrical access to
the cell from below the PPC plate. Once access to the cell is
obtained, experimental recordings are initiated. Two types of
electrodes, a common ground electrode below the plate and separate
recording electrodes dipped into each well, allow for control of
the voltage (the potential across the cell membrane) and for
recording the flow of ionic current across the membrane of the
entire cell. The IonWorks Quattro system is a semi-automated patch
clamp workstation that allows recordings to be made from 384 wells.
Dispensing of cells to the patch plate, seal formation and
electrical access are automated while compound delivery is
performed by computer-controlled fluid handlers from 384-well
compound plates.
[0463] The following description illustrates the IonWorks screening
assay to identify compounds that modulate (activate or block)
TRPML3 function. For a well in a plate to provide data two criteria
must be met: (i) initially, the well must have most of its holes
open (the average resistance across the 64 holes must be >1 mOhm
and <10 mOhm) and (ii) after cell addition, most holes must have
a cell forming a high mOhm seal with the patch plate (the average
resistance across the 64 holes must be >10 mOhm). If these two
criteria are met, the instrument collects data from pre-compound
and post-compound scans. In the example shown (FIG. 28), 94% of
wells have a resistance greater than 10 mOhm. In each scan currents
are measured as the voltage is modified (see example voltage
command trace in FIG. 29).
[0464] Since TRPML3 channels exhibit inward rectification, the
recordings will show large inward currents at hyperpolarized
potentials and small outward currents at depolarized potentials
(FIG. 29). Compounds will be applied at concentrations between
.about.1 uM and 100 uM. If the compound functions as a TRPML3
enhancer, the current passing through TRPML3 channels in the cell
membrane increases. If the compound functions as a TRPML3 blocker,
the current passing through TRPML3 channels in the membrane
decreases. TRPML3 currents can be examined at two different
voltages: for example, -120 mV and -40 mV. Compounds that enhance
TRPML3 by affecting the voltage dependent rectification will be
preferentially detected at -40 mV where the rectification is strong
while compounds that block TRPML3 will be preferentially detected
at -120 mV where the TRPML3 currents are larger. To quantitate the
effect of a compound on TRPML3 function, we use the following
formula: [(A-Ao)/(B-Bo)].times.100. B and Bo are the currents
measured before compound addition while A and Ao are the currents
measured after compound addition. A and B are the currents at the
test voltage (either -120 mV or -40 mV) while Ao or Bo is the
current at 0 mV. This value leads to a % modulation factor that is
used to gauge the activity of compounds in our assay. For example,
if the % modulation factor is equal to 200%, then the compound
doubles TRPML3 activity compared to control values in the absence
of compound. If the % modulation factor is equal to 50%, then the
compound decreases TRPML3 activity by one-half over basal control
values (in the absence of compound).
[0465] Negative control experiments are performed in parental cells
to demonstrate that effects observed with compounds in TRPML3
expressing cells are due to currents flowing through TRPML3
channels and not due to currents flowing through channels
endogenously expressed in the cell membrane. Compounds specifically
modulating TRPML3 should not affect currents in control CHO-K1
cells and should exhibit % modulation factors near 100%.
[0466] More complex analyses are performed on compounds displaying
large % modulation factors and having no effect on control CHO-K1
cells. The assays include current/voltage (I/V) curves, GdCl3
competition experiments (GdCl3 is a blocker of TRPML3), and
dose-response curves. For I/V curves, currents are measured in
voltage steps from -120 to +60 mV, in 10 mV increments in the
presence and absence of compound, to investigate the magnitude of
compound modulation. The slope of the I/V curve is indicative of
the magnitude of current modulation by the compound of interest.
Strong enhancers increase the slope of I/V curves, indicative of
increased opening of TRPML3 ion channels. Strong blockers decrease
the slope of I/V curves, indicative of increased closing of TRPML3
ion channels. Control I/V curves performed in the presence of
compound should be identical and superimposable with I/V curves
performed in the absence of compound.
[0467] GdCl3 competition experiments are performed to demonstrate
that compound effects are TRPML3 dependent. First, compound is
applied to determine the % modulation factor then a saturating dose
of GdCl3 (or some other TRPML3 blocker) is applied. For an enhancer
to work directly on the TRPML3 channel, currents from cells treated
with enhancer plus GdCl3 should resemble currents seen for cells
treated only with GdCl3. This experiment shows that when the
channel is blocked the compound does not have an enhancing effect;
therefore, the compound must directly modulate TRPML3 channel
function.
[0468] Dose-response curves are performed to determine the
concentration at which the compound exhibits half-maximal activity
(EC50 for enhancers and IC50 for blockers). The lower the EC50 or
IC50 value, the more active the compound is as a TRPML3 modulator.
Dose-response curves are performed by sequentially applying
increasing concentrations of compound starting from low doses
(.about.1 nM) and progressing to high doses (.about.1 mM). %
modulation factors are calculated as described above and plotted as
a function of compound concentration on a logarithmic scale to
determine an EC50 or IC50 value for the compound.
[0469] A flowchart illustrating the sequence of experiments
performed to examine the effect of a compound on TRPML3 function is
depicted in FIG. 30, including screening at a holding potential of
-120 mV and -40 mV, I/V curves, GdCl3 competition tests,
dose-response curves, and negative control experiments.
[0470] Animal Models
[0471] Animal models also find potential use in screening for
modulators of gene activity. Transgenic animal technology results
in gene overexpression, whereas siRNA and gene knockout technology
results in absent or reduced gene expression following homologous
recombination with an appropriate gene targeting vector. The same
technology can also be applied to make knock-out cells. When
desired, tissue-specific expression or knockout of the target gene
may be necessary. Transgenic animals generated by such methods find
use as animal models of responses related to the gene target. For
example such animals expressing a gene or genes according to the
invention may be used to derive supertaster phenotypes such as for
use in screening of chemical and biological toxins,
rancid/spoiled/contaminated foods, and beverages or for screening
for therapeutic compounds that modulate taste stem cell
differentiation.
[0472] Knock-out cells and transgenic mice can be made by insertion
of a marker gene or other heterologous gene into an endogenous gene
site in the mouse genome via homologous recombination. Such mice
can also be made by substituting an endogenous gene with a mutated
version of the target gene, or by mutating an endogenous gene,
e.g., by exposure to known mutagens.
[0473] A DNA construct is introduced into the nuclei of embryonic
stem cells. Cells containing the newly engineered genetic lesion
are injected into a host mouse embryo, which is re-implanted into a
recipient female. Some of these embryos develop into chimeric mice
that possess germ cells partially derived from the mutant cell
line. Therefore, by breeding the chimeric mice it is possible to
obtain a new line of mice containing the introduced genetic lesion
(see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric
targeted mice can be derived according to Hogan et al.,
Manipulating the Mouse Embryo: A Laboratory Manual (1988) and
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach
(Robertson, ed., 1987).
[0474] Preferred Animal Model Assay Embodiment--Varitint-Waddler
Mice Studies
[0475] This invention also contemplates the use of a mouse model,
termed the Varitint waddler mouse, or similar transgenic animals
where TRPML3 salty taste cells are specifically ablated from taste
buds and where salty taste is greatly diminished to study the
effect of TRPML3 in vivo on salty taste and other functions
relating to sodium metabolism as well as the use of this gene
mutation in creating animals depleted in specific cell types such
as salty taste cells, melanocytes, pituitary cells, and adrenal
cells.
[0476] The Varitint waddler mouse has a gain of function A419P
mutation in the TRPML3 ion channel (Di Palma et al PNAS 99(23):
14994-14999, 2002) that arose from a spontaneous mutation in 1942
(Cloudman et al J. Heredity 36: 258-263, 1945). These mice are
termed Varitint waddler due to their two most obvious phenotypes: a
variegated coat color (variable tint of fur) and deficiency in the
vestibular system (circling behavior and waddling that resembles a
duck). The increased activity of A419P TRPML3 alters the ionic
equilibrium of cells expressing TRPML3, including melanocytes in
the skin as well as hair cells in the inner ear and vestibular
system, and results in death of these cell populations (Xu et al.
PNAS 104(46): 18321-18326, 2007; Grimm et al. PNAS 104(49):
19583-19588, 2007; Nagata et al. PNAS 105(1): 353-358, 2008; Kim et
al. J. Biol. Chem. 282(50): 36138-36142, 2007). Cell death is
likely attributable to uncontrolled entry of sodium and/or calcium
ions into the cytoplasm. Thus, the Varitint waddler mouse is a
model for cell ablation where cells expressing A419P TRPML3 die
off.
[0477] Since TRPML3 is specifically expressed in taste cells on the
tongue, experiments were performed to determine if taste cells
expressing TRPML3 were ablated in the Varitint waddler mouse.
TRPML3 is not detectable in purified taste cells from Varitint
waddler mice using end-point PCR (FIG. 31) or real-time
quantitative PCR (FIG. 32). By contrast, expression of genes
involved in sweet, bitter, umami, and sour taste are similar in
Varitint waddler and wild-type control mice (FIGS. 31-32). FIG. 33
illustrates that TRPM5 (sweet, bitter, umami, GPR113) and
PKD2L1/PKD1L3 (sour) taste cells are intact in Varitint waddler
mice using the histological in situ hybridization technique.
Therefore, taste cell populations that do not express TRPML3
(including sweet, bitter, umami, GPR113, and sour) are unaffected
in the Varitint waddler mouse model. Therefore, Varitint waddler
mice contain taste buds lacking TRPML3 taste cells and Varitint
waddler mice can be used to study salty taste in the absence of
this cell population.
[0478] In addition, electrophysiological CT nerve recordings are an
established method to study taste biology in rodent systems and
have been used to elucidate the effect of genetic mutations on
physiological responses to diverse taste stimuli (Damak et al,
Science. 2003 301(5634):850-3; Lyall et al, J. Physiol. 2004 558(Pt
1):147-59; Zhao et al, Cell. 2003 115(3):255-66; Mueller et al,
Nature. 2005 434(7030):225-9). The CT nerve innervates the anterior
tongue encompassing taste buds in fungiform and some foliate taste
papilla; thus, CT nerve activity represents a measure of taste
receptor cell function in response to tastant application to the
front of the tongue. CT nerve recording methodology was carried out
as previously described (Lyall et al, J. Physiol. 2004 558(Pt
1):147-59; Treesukosol et al, Am J Physiol Regul Integr Comp
Physiol. 2007 May; 292(5):R1799-809; Dahl et al, Brain Research.
1997 756:22-34), using procedures familiar to those skilled in the
art.
[0479] Using CT nerve recordings, Varitint waddler mice were shown
to exhibit a deficiency in the response to sodium chloride.
Specifically, Varitint waddler mice have a greatly reduced
benzamil-insensitive CT nerve response to sodium chloride (FIG.
34). Since TRPML3 is not blocked by amiloride or the amiloride
analog benzamil, the benzamil-insensitive CT response is largely
attributable to TRPML3. Both the initial (phasic) and sustained
(tonic) components of the CT nerve response were attenuated in
Varitint waddler mice. These data indicate that elimination of
TRPML3 taste cells substantially reduces the ability of mice to
taste salt, and point to a central role of TRPML3 taste cells as
professional salty taste cells.
[0480] These results further show that the Varitint waddler mice
have taste buds in which TRPML3 taste cells are specifically
ablated and that these mice can be used in taste studies wherein
salty taste is specifically affected.
[0481] Also, these results show that Varitint waddler mice exhibit
a deficiency in the benzamil-insensitive CT nerve response to
sodium chloride and Varitint waddler mice exhibit a deficiency in
the initial (phasic) and sustained (tonic) components of the CT
nerve response to sodium chloride.
[0482] Importantly, these results show that the expression of A419P
TRPML3 can be used in order to specifically ablate cell types and
create mouse model systems lacking different cell populations.
[0483] Also, these animals can be used to study the effect of A419P
TRPML3 as a toxin to kill specific cell types.
[0484] Candidate Modulators
[0485] The compounds tested as modulators of the putative taste
related proteins or other non-taste related functions and
phenotypes involving taste cells can be any small organic molecule,
or a biological entity, such as a protein, e.g., an antibody or
peptide, a sugar, a nucleic acid, e.g., an antisense
oligonucleotide or a ribozyme, or a lipid. Alternatively,
modulators can be genetically altered versions of a protein.
Typically, test compounds will be small organic molecules,
peptides, lipids, and lipid analogs. In one embodiment, the
compound is a menthol analog, either naturally occurring or
synthetic.
[0486] Essentially any chemical compound can be used as a potential
modulator or ligand in the assays of the invention, although most
often compounds can be dissolved in aqueous or organic (especially
DMSO-based) solutions are used. The assays are designed to screen
large chemical libraries by automating the assay steps and
providing compounds from any convenient source to assays, which are
typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays). It will be appreciated that
there are many suppliers of chemical compounds, including Sigma
(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St.
Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland)
and the like.
[0487] In one preferred embodiment, high throughput screening
methods involve providing a combinatorial small organic molecule or
peptide library containing a large number of potential therapeutic
compounds (potential modulator or ligand compounds). Such
"combinatorial chemical libraries" or "ligand libraries" are then
screened in one or more assays, as described herein, to identify
those library members (particular chemical species or subclasses)
that display a desired characteristic activity. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics.
[0488] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0489] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication No. WO
93/20242), random bio-oligomers (e.g., PCT Publication No. WO
92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),
diversomers such as hydantoins, benzodiazepines and dipeptides
(Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)),
vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.
114:6568 (1992)), nonpeptidal peptidomimetics with glucose
scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218
(1992)), analogous organic syntheses of small compound libraries
(Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates
(Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates
(Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid
libraries (see Ausubel, Berger and Sambrook, all supra), peptide
nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),
antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,
14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries
(see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S.
Pat. No. 5,593,853), small organic molecule libraries (see, e.g.,
benzodiazepines, Baum C&EN, January 18, page 33 (1993);
isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and
metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat.
Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No.
5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the
like).
[0490] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, P A, Martek
Biosciences, Columbia, Md.). C. Solid State and Soluble High
Throughput Assays
[0491] Additionally soluble assays can be affected using a target
taste specific protein, or a cell or tissue expressing a target
taste protein disclosed herein, either naturally occurring or
recombinant. Still alternatively, solid phase based in vitro assays
in a high throughput format can be effected, where the protein or
fragment thereof, such as the cytoplasmic domain, is attached to a
solid phase substrate. Any one of the assays described herein can
be adapted for high throughput screening, e.g., ligand binding,
calcium flux, change in membrane potential, etc.
[0492] In the high throughput assays of the invention, either
soluble or solid state, it is possible to screen several thousand
different modulators or ligands in a single day. This methodology
can be used for assaying proteins in vitro, or for cell-based or
membrane-based assays comprising a protein. In particular, each
well of a microtiter plate can be used to run a separate assay
against a selected potential modulator, or, if concentration or
incubation time effects are to be observed, every 5-10 wells can
test a single modulator. Thus, a single standard microtiter plate
can assay about 100 (e.g., 96) modulators. If 1536 well plates are
used, then a single plate can easily assay from about 100-about
1500 different compounds. It is possible to assay many plates per
day; assay screens for up to about 6,000, 20,000, 50,000, or more
than 100,000 different compounds are possible using the integrated
systems of the invention.
[0493] For a solid state reaction, the protein of interest or a
fragment thereof, e.g., an extracellular domain, or a cell or
membrane comprising the protein of interest or a fragment thereof
as part of a fusion protein can be bound to the solid state
component, directly or indirectly, via covalent or non covalent
linkage e.g., via a tag. The tag can be any of a variety of
components. In general, a molecule which binds the tag (a tag
binder) is fixed to a solid support, and the tagged molecule of
interest is attached to the solid support by interaction of the tag
and the tag binder.
[0494] A number of tags and tag binders can be used, based upon
known molecular interactions well described in the literature. For
example, where a tag has a natural binder, for example, biotin,
protein A, or protein G, it can be used in conjunction with
appropriate tag binders (avidin, streptavidin, neutravidin, the Fc
region of an immunoglobulin, etc.) Antibodies to molecules with
natural binders such as biotin are also widely available and
appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue
SIGMA, St. Louis Mo.).
[0495] Similarly, any haptenic or antigenic compound can be used in
combination with an appropriate antibody to form a tag/tag binder
pair. Thousands of specific antibodies are commercially available
and many additional antibodies are described in the literature. For
example, in one common configuration, the tag is a first antibody
and the tag binder is a second antibody which recognizes the first
antibody. In addition to antibody-antigen interactions,
receptor-ligand interactions are also appropriate as tag and
tag-binder pairs. For example, agonists and antagonists of cell
membrane receptors (e.g., cell receptor-ligand interactions such as
transferring, c-kit, viral receptor ligands, cytokine receptors,
chemokine receptors, interleukin receptors, immunoglobulin
receptors and antibodies, the cadherin family, the integrin family,
the selectin family, and the like; see, e.g., Pigott & Power,
The Adhesion Molecule Facts Book 1 (1993). Similarly, toxins and
venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),
intracellular receptors (e.g. which mediate the effects of various
small ligands, including steroids, thyroid hormone, retinoids and
vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both
linear and cyclic polymer configurations), oligosaccharides,
proteins, phospholipids and antibodies can all interact with
various cell receptors.
[0496] Synthetic polymers, such as polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, and polyacetates
can also form an appropriate tag or tag binder. Many other tag/tag
binder pairs are also useful in assay systems described herein, as
would be apparent to one of skill upon review of this
disclosure.
[0497] Common linkers such as peptides, polyethers, and the like
can also serve as tags, and include polypeptide sequences, such as
poly gly sequences of between about 5 and 200 amino acids. Such
flexible linkers are known to persons of skill in the art. For
example, poly(ethylene glycol) linkers are available from
Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally
have amide linkages, sulfhydryl linkages, or heterofunctional
linkages.
[0498] Tag binders are fixed to solid substrates using any of a
variety of methods currently available. Solid substrates are
commonly derivatized or functionalized by exposing all or a portion
of the substrate to a chemical reagent which fixes a chemical group
to the surface which is reactive with a portion of the tag binder.
For example, groups which are suitable for attachment to a longer
chain portion would include amines, hydroxyl, thiol, and carboxyl
groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to
functionalize a variety of surfaces, such as glass surfaces. The
construction of such solid phase biopolymer arrays is well
described in the literature. See, e.g., Merrifield, J. Am. Chem.
Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,
e.g., peptides); Geysen et al., J. Immunol. Meth. 102:259-274
(1987) (describing synthesis of solid phase components on pins);
Frank & Doring, Tetrahedron 44:6031-6040 (1988) (describing
synthesis of various peptide sequences on cellulose disks); Fodor
et al., Science, 251:767-777 (1991); Sheldon et al., Clinical
Chemistry 39(4):718-719 (1993); and Kozal et al., Nature Medicine
2(7):753-759 (1996) (all describing arrays of biopolymers fixed to
solid substrates). Non-chemical approaches for fixing tag binders
to substrates include other common methods, such as heat,
cross-linking by UV radiation, and the like.
[0499] Having described the invention supra, the examples provided
infra further illustrate some preferred embodiments of the
invention. These examples are provided only for purposes of
illustration and should not be construed as limiting the subject
invention.
[0500] Practical Applications of the Invention
[0501] Compounds which modulate, preferably enhance the activity of
taste specific genes identified according to the invention have
important implications in modulation of human salty taste and
potentially other taste modalities or taste in general. In addition
these compounds are potentially useful in therapeutic applications
involving other taste cell related functions and phenotypes such as
taste cell turnover, digestive diseases, digestive function,
regulation of metabolism, regulation of immunity in the oral cavity
and/or digestive system and the like.
[0502] Compounds which activate taste ion channels in taste
papillae on the tongue can be used to enhance salt sensation by
promoting Na.sup.+ transport into taste bud cells. This has obvious
consumer applications in improving the taste and palatability of
low salt foods and beverages.
[0503] In addition the genes and gene products herein can be used
as markers for identifying, isolating or enriching specific taste
cell types or lineages.
[0504] Further the genes and gene products specific to taste cells
identified herein can be used to identify compounds that modulate
apoptosis of taste cells, modulate transcription factors that
control taste receptor expression, modulate autocrine/paracrine
modulation of taste cell development, prolong taste bud lifetime,
yield supertaster animal phenotypes for use in screening such as
for bioterrorism or animals for use in screening for compounds that
induce the activation and differentiation of stem cells into taste
cells in vivo.
[0505] Also the subject genes, gene products and cells which
express same may be used to screen for compounds that affect
trafficking of taste receptors to and from the apical
membrane/taste pore region to enhance or repress general or
specific tastes, regulation of taste cell action potential firing
frequency/membrane potential to control the intensity of general or
specific tastes, regulation of neurotransmitter release to afferent
nerve to control the intensity of general or specific taste, and
autocrine/paracrine modulation of taste receptor function.
[0506] Further the subject genes, gene products and cells which
express same can be used to identify compounds that regenerate
taste cells such as in geriatric individuals or patients with
cancer, chemotherapy radiation, injury or surgery affecting taste,
drug-induced dysgeusia, ageusia, and for alleviating taste bud
loss.
[0507] Still further the subject genes and gene products and cells
which express same can be used to screen for compounds that affect
oral hygiene, halitosis, detoxification of noxious substances in
the oral cavity, and neutralization/elimination of bacteria,
viruses, and other immunogens in the saliva/mouth or digestive
tract.
[0508] Preferably, modulators of TRPML3 can be used as flavor
additives in order to elicit or modulate (enhance or inhibit) salty
taste perception, and to treat conditions and physiological
functions involving sodium metabolism, absorption and excretion. In
particular, modulators of TRPML3 can be added to foods, beverages
and medicaments and other consumer products in order to modulate or
mask the salty taste thereof. In addition, TRPML3 modulators and
enhancers can be used to treat and modulate TRPML3 related cardiac
and urinary functions such as blood pressure, fluid retention,
urine production, stroke, heart attack, arrhythmias, aldosterone
production, and vasopressin release.
[0509] In addition, transgenic TRPML3 animals, e.g., knockout and
knockin animals have practical applications in the study of the
effects of sodium metabolism and other activities on physiological
processes and diseases such as Addison's disease as well as for the
identification of compounds that modulate TRPML3.
[0510] The following examples relating to TRPML3 provide
confirmatory evidence suggesting that TRPML3 encodes a salty taste
receptor polypeptide and were affected using the materials and
methods described supra. These examples are put forth so as to
provide those of ordinary skill in the art with a complete
disclosure and description of how to make and use the subject
invention, and are not intended to limit the scope of what is
regarded as the invention.
EXAMPLES
Example 1
[0511] This example relates to the experiments and molecular
biology data which are contained in FIG. 1 that show that TRPML3 is
a taste-specific gene. RT-PCR of human (left) and monkey (right)
taste buds (taste) and lingual epithelial cells (lingual) collected
by laser capture microdissection was affected. FIG. 1 shows that
TRPML3 is only expressed in taste cells, similar to the known
taste-specific genes T1R2 and TRPM5. The Figure also shows that the
housekeeping gene beta-actin is expressed in both taste and lingual
cells demonstrating that RNA from both samples is of high quality.
`+` indicates reverse transcription was performed and `-` indicates
that no reverse transcription was performed (negative control).
Bands are only observed with reverse transcription. All bands were
cloned and sequenced to confirm gene identities.
Example 2
[0512] This example contains the electrophysiological assays
contained in FIG. 2 which reveal that TRPML3 forms a sodium
channel. Whole cell patch clamp electrophysiology of cells
expressing human TRPML3 was affected as depicted therein. It can be
seen that TRPML3 generates a sodium leak current that is blocked
upon removal of sodium and replacement with the large impermeant
cation NMDG. The top trace in the same Figure shows current at a
holding potential of -60 mV. The middle traces in FIG. 2 show
current-voltage traces from -100 mV to +60 mV in the presence
(NaCl) and absence (NMDG-Cl) of sodium. The bottom graph in the
Figure shows current voltage curves in the presence (dark blue
line; diamonds) and absence (magenta line; squares) of sodium. It
can be seen that TRPML3 exhibits inward rectification (more current
at negative voltages compared to positive voltages).
Example 3
[0513] This example relates to the electrophysiological assays the
results of which are contained in FIG. 3. These results obtained
using the human TRPML3 channel properties are consistent with human
salty taste psychophysics. The top graph in the Figure contains
current-voltage curves showing TRPML3 sodium conductance (dark blue
line; diamonds) is not blocked by 30 uM amiloride (magenta line;
squares). Both human salty taste and TRPML3 are not blocked by
amiloride. The bottom graph in the same figure contains
current-voltage curves showing TRPML3 is equally permeable to the
salty cations sodium (dark blue line; diamonds) and lithium
(magenta line; squares). This is consistent with TRPML3 encoding a
human salty taste receptor since sodium and lithium are equally
salty to humans and both cations permeate the human TRPML3
channel.
Example 4
[0514] This example relates to the immunohistochemical labeling
experiments contained in FIG. 4. It can be seen therein that the
TRPML3 protein is expressed in the apical membrane region near the
taste pore. Particularly, it is seen that the TRPML3 antibody
labels taste cell processes extending to the taste pore (left
image). In addition magnification of the apical taste bud domain
facing the saliva clearly demonstrates that the TRPML3 protein is
expressed at the taste pore region (3 right images; taste pore
denoted with blue arrows). This location is ideally suited for
TRPML3 to sense sodium in the saliva. Similar to TRPML3, other
taste receptors (sweet, bitter, umami, and sour) are also polarized
to the taste pore where they sample saliva for their requisite
tastants
Example 5
[0515] This example relates to the immunochemical labeling
experiments contained in FIG. 5. These results show that the TRPML3
protein is not expressed in TRPM5 cells. Specifically, double label
immunohistochemistry with TRPM5 (green; left images) and TRPML3
(red; middle images) in monkey CV papilla was effected. It can be
seen from the figure that cells expressing TRPM5 and TRPML3 are
distinct (merged images on the right). These data indicate that
TRPML3 is not expressed in TRPM5 cells (encompassing sweet, bitter,
and umami cells) but only in professional salty taste cells.
Example 6
[0516] This example relates to the immunohistochemical labeling
experiments contained in FIG. 6. These results show that the TRPML3
protein is not expressed in PKD2L1 cells. Double label
immunohistochemistry was effected with PKD2L1 (green; left images)
and TRPML3 (red; middle images) in monkey CV papilla. It can be
seen from the figure that the cells expressing PKD2L1 and TRPML3
are distinct (merged images on the right). These data indicate that
TRPML3 is not expressed in PKD2L1 cells (encompassing sour cells)
but in professional salty taste cells.
Example 7
[0517] I/V curves in oocytes injected with human TRPML3 cRNA.
[0518] The following example illustrates the oocyte screening assay
to identify compounds that modulate (activate or block) TRPML3
function. Oocytes expressing TRPML3 are identified by I/V curve
analysis and sodium-replacement with NMDG. See FIG. 7. Currents are
measured in voltage steps from -100 to +60 mV, in 10 mV increments.
Since TRPML3 channels exhibit inward rectification, oocytes with
large inward currents at hyperpolarized potentials and small
outward currents at depolarized potentials express TRPML3 channels.
NMDG is a large cation that cannot permeate TRPML3. Thus, current
inhibited by NMDG represents the TRPML3-dependent sodium current.
Inhibition of current with NMDG is used as another internal control
to verify that the oocytes express functional TRPML3 protein.
[0519] The inwardly rectifying I/V curves, denoted by more current
at hyperpolarized potentials (more negative potentials) and less
current at depolarized potentials (more positive potentials),
indicate that oocytes express TRPML3 ion channels. Replacement of
sodium with NMDG blocks TRPML3 current. Subtraction of I/V curves
in sodium and NMDG yields the TRPML3 specific sodium current.
Diamonds (.diamond-solid.) denote I/V curve in sodium (NaCl)
solution; squares (.box-solid.) denote I/V curve in sodium-free
NMDG solution; triangles (.tangle-solidup.) denote subtraction of
NaCl and NMDG I/V curves and represents the TRPML3-specific sodium
current.
Example 8
[0520] Screening oocytes injected with human TRPML3 cRNA for
compounds that may modulate TRPML3 activity.
[0521] A compound at a concentration between .about.1 uM and
.about.100 uM is applied to oocytes exhibiting TRPML3 function. See
FIG. 2. If the compound functions as a TRPML3 enhancer, the current
passing through TRPML3 channels in the oocyte membrane increases
(becomes more negative). If the compound functions as a TRPML3
blocker, the current passing through TRPML3 channels in the oocyte
membrane decreases (becomes less negative). To quantitate the
effect of a compound on TRPML3 function, the following formula is
used:
[(A-Ao)/(B-Bo)]x-100
[0522] A is the current following compound treatment, Ao is the
current preceding compound treatment, B is the current following
NMDG treatment, and Bo is the current preceding NMDG treatment.
This value leads to a % modulation factor that is used to gauge the
activity of compounds in the assay. For example, if the %
modulation factor is equal to 100%, then the compound increases
TRPML3 activity 100% over basal control values (in the absence of
compound). If the % modulation factor is equal to -100%, then the
compound decreases TRPML3 activity 100% over basal control values
(in the absence of compound). % modulation factors are calculated
individually for each of the oocytes in the OpusXpress system and
then an average and standard deviation are determined for each
compound.
[0523] Negative control experiments are performed in oocytes not
injected with TRPML3 cRNA to demonstrate that effects observed with
compounds in TRPML3 expressing oocytes are due to currents flowing
through TRPML3 channels and not due currents flowing through
channels endogenously expressed in the oocyte membrane. Compounds
specifically modulating TRPML3 should not affect currents in
uninjected oocytes and should exhibit % modulation factors near
zero.
[0524] As illustrated in FIG. 8, for each compound screened, a %
modulation factor is calculated. This value corresponds to the
magnitude of the current change due to compound divided by the
magnitude of the current change due to NMDG multiplied by -100%. In
this example, three compounds (100 uM) are screened in succession
in four, out of a possible maximum eight, oocytes voltage clamped
to -60 mV in the OpusXpress system. All four oocytes expressed
TRPML3, as evidenced by the inhibitory effect of NMDG on measured
oocyte currents. All three compounds did not significantly modulate
TRPML3 function as currents were similar before and after compound
addition.
Example 9
[0525] I/V curves with the TRPML3 blocker gadolinium.
[0526] More complex analyses are performed on compounds displaying
large % modulation factors and having no effect on oocytes not
injected with TRPML3 cRNA. The assays include current/voltage (I/V)
curves, NMDG competition experiments, and dose-response curves. For
I/V curves, currents are measured in voltage steps from -100 to +60
mV, in 10 mV increments in the presence and absence of compound, to
investigate the magnitude of compound modulation. The slope of the
I/V curve is indicative of the magnitude of current modulation by
the compound of interest. Strong enhancers increase the slope of
I/V curves, indicative of increased opening of TRPML3 ion channels.
Strong blockers decrease the slope of I/V curves, indicative of
increased closing of TRPML3 ion channels. FIG. 9 illustrates how
gadolinium blocks TRPML3 and decreases the slope of the I/V curve.
In oocytes not injected with TRPML3 cRNA, I/V curves performed in
the presence of compound should be identical and superimposable
with I/V curves performed in the absence of compound. Small squares
illustrate an I/V curve in sodium solution and large squares
illustrate an I/V curve in 300 uM gadolinium (GdCl.sub.3) solution.
Note that gadolinium blocks TRPML3 current and decreases the slope
of the I/V curve.
Example 10
[0527] NMDG competition experiments
[0528] NMDG competition experiments are performed to demonstrate
that compound effects are TRPML3 dependent. First, NMDG is applied
to demonstrate TRPML3 expression in the oocytes. Then, compound is
applied to determine the % modulation factor. Finally, NMDG and
compound are co-applied. For an enhancer to work directly on the
TRPML3 channel, co-application of NMDG plus compound should exhibit
a NMDG-type response, meaning that currents are inhibited and not
enhanced. This experiment shows that when sodium is absent, and
replaced with the non-permeant cation NMDG, the compound cannot
have an enhancing effect; therefore, the compound must directly
modulate TRPML3 sodium channel function.
Example 11
[0529] Dose-response curves
[0530] Dose-response curves are performed to determine the
concentration at which the compound exhibits half-maximal activity
(EC50 for enhancers and IC50 for blockers). The lower the EC50 or
IC50 value, the more active the compound is as a TRPML3 modulator.
Dose-response curves are performed by sequentially applying
increasing concentrations of compound starting from low doses
(.about.1 nM) and progressing to high doses (.about.1 mM). %
modulation factors are calculated as described above and plotted as
a function of compound concentration on a logarithmic scale to
determine an EC50 or IC50 value for the compound.
Example 12
[0531] Expression of constitutively active sodium channels increase
basal fluorescence in cells loaded with specific membrane potential
dyes.
[0532] HEK293 cells were transiently transfected with RFP,
.alpha.ENaC or A419P-TRPML3 and were loaded with a membrane
potential dye (R-8034; Molecular Devices) in HBSS at room
temperature for 30 minutes. Membrane potential (fluorescent signal)
was monitored on a FLIPR system (Molecular Devices). See FIG. 11.
Results show that cells expressing A419P-TRPML3 and .alpha.ENaC
show an elevated basal fluorescence when compared to RFP
transfected cells (RFP is a control vector). These results mean
that the constitutive activity of A419P-TRPML3 and .alpha.ENaC
causes the cell membrane to be more depolarized and that we can
measure activity of A419P-TRPML3 in this FLIPR assay.
Example 13
[0533] Application of gadolinium reduces the increase in basal
fluorescence in cells expressing A419P-TRPML3.
[0534] HEK293 cells were transiently transfected with RFP or
A419P-TRPML3 and were loaded with a membrane potential dye (R-8034;
Molecular Devices) in HBSS at room temperature for 30 minutes. See
FIG. 12. Membrane potential (fluorescent signal) was monitored on a
FLIPR system (Molecular Devices). Results show that addition of
gadolinium (large, short arrowhead) significantly reduces the basal
fluorescence close to values obtained in RFP-transfected cells
(long arrow and trace 2). These results indicate that both the
constitutive activity of A419P-TRPML3 and the activity of a TRPML3
modulator can be detected in this assay.
Example 14
[0535] Application of gadolinium reduces the increase in basal
fluorescence in cells expressing A419P-TRPML3 in a dose-dependent
fashion.
[0536] HEK293 cells were transiently transfected with RFP or
A419P-TRPML3 and were loaded with a membrane potential dye (R-8034;
Molecular Devices) in HBSS at room temperature for 30 minutes.
HBSS, NMDG and increasing concentrations of gadolinium were added
to the cells and resulting changes in membrane potential
(fluorescent signal) was monitored on a FLIPR system (Molecular
Devices). See FIG. 13. Results show that increasing concentration
of gadolinium significantly reduces the basal fluorescence to a
much greater extent to the effect observed in RFP-transfected
cells.
Example 15
[0537] Titration of TRPML3 plasmid.
[0538] HEK293 cells were transiently transfected with RFP (0 ug) or
increasing amounts of A419P-TRPML3 plasmid (from 0.02 ug to 2 ug)
and were loaded with a membrane potential dye (R-8034; Molecular
Devices) in HBSS at room temperature for 30 minutes. See FIG. 4.
HBSS (control) and 4 mM gadolinium were added to the cells and
resulting changes in membrane potential (fluorescent signal) was
monitored on a FLIPR system (Molecular Devices). Results show that
increasing the amount of TRPML3 plasmid up to 0.5 g increases the
size of the gadolinium effect.
Example 16
[0539] Effect of gadolinium is specific for TRPML3.
[0540] HEK293 cells were transiently transfected with RFP,
A419P-TRPML3 or .alpha.ENaC plasmid and were loaded with a membrane
potential dye (R-8034; Molecular Devices) in HBSS at room
temperature for 30 minutes. HBSS (control), 3 mM gadolinium and 30
uM Amiloride were added to the cells and resulting changes in
membrane potential (fluorescent signal) was monitored on a FLIPR
system (Molecular Devices). See FIG. 15. Results show that
gadolinium preferentially reduces basal fluorescence counts in
A419P-TRPML3 transfected cells while amiloride preferentially
reduces basal fluorescence counts in .alpha.ENaC-transfected
cells.
Example 17
[0541] Transducing HEK293 cells with baculovirus encoding
A419P-TRPML3 doubles the assay window.
[0542] HEK293 cells were transduced with a modified baculovirus
allowing expression of A419P-TRPML3 in mammalian cells (BacMaM).
After 24 hours infected cells were loaded with a membrane potential
dye (R-8034; Molecular Devices) in HBSS at room temperature for 30
minutes. HBSS (control), 2 mM and 3 mM gadolinium were added to the
cells and resulting changes in membrane potential (fluorescent
signal) was monitored on a FLIPR system (Molecular Devices). See
FIG. 16.
Example 17
[0543] Example of screening data obtained with A419P-TRPML3
expressing cells.
[0544] HEK293 cells were transduced with a modified baculovirus
allowing expression of A419P-TRPML3 in mammalian cells (BacMaM).
After 24 hours infected cells were loaded with a membrane potential
dye (R-8034; Molecular Devices) in HBSS at room temperature for 30
minutes. See FIG. 17. 320 different compounds (red dots), HBSS
(black dots) and gadolinium (blue dots) were added to the cells,
from a 384 well compound plate, and resulting changes in membrane
potential (fluorescent signal) was monitored on a FLIPR system
(Molecular Devices). In this experiment two primary hits that
apparently block A419P-TRMPL3 were identified.
Example 18
[0545] Summary of a 10,000 compound miniscreen with A419P-TRPML3
expressing cells.
[0546] HEK293 cells were transduced with a modified baculovirus
allowing expression of A419P-TRPML3 in mammalian cells (BacMaM).
After 24 hours infected cells were loaded with a membrane potential
dye (R-8034; Molecular Devices) in HBSS at room temperature for 30
minutes and treated as described in FIG. 17. See FIG. 18. 10,000
compounds were screened and several primary hits were identified,
including 52 blocker hits and 113 enhancer hits.
[0547] Examples 11-18 show that under these experimental
conditions, cells expressing A419P-TRPML3 showed a significant
increase in basal fluorescence relative to cells transfected with a
control vector (RFP). Gadolinium, a blocker of TRP channels,
reversed the increase in fluorescence in a dose-dependent and
specific fashion. Amiloride, a blocker of .alpha.ENaC, had no
effect on the increased basal fluorescence elicited by expression
of A419P-TRPML3. Several thousand compounds have now been screened
in this assay and several hits have been identified. These hits
will be further evaluated by electrophysiology and taste tests.
Example 19
[0548] Construction of Codon Optimized TRPML3 Gene and Mutant
[0549] As shown in FIG. 19 a codon optimized TRPML3 gene was
constructed. Codons comprise three nucleotides that encode a
specific amino acid in a protein sequence. Since there are 61
different codon nucleotide triplets that encode 20 amino acids,
most amino acids can be encoded by more than one codon. Codon
optimization, the use of the favored codon for each amino acid in a
particular species, can improve the functional expression of
proteins by increasing the speed and accuracy of translation
without changing the protein sequence.
[0550] The inventors used a codon-optimized version of the human
TRPML3 gene that is 76.4% homologous to non-codon optimized TRPML3
at the DNA level (FIG. 19). Codons were optimized for optimal
translation of human sequences. An active form of TRPML3 (A419P
TRPML3) was also generated by mutating alanine 419 to proline in
the 5.sup.th transmembrane domain. This mutation results in TRPML3
channels that are in an open confirmation (Xu et al. PNAS 104(46):
18321-18326, 2007; Grimm et al. PNAS 104(49): 19583-19588, 2007;
Nagata et al. PNAS 105(1): 353-358, 2008; Kim et al. J. Biol. Chem.
282(50): 36138-36142, 2007); therefore, A419P TRPML3 is
particularly useful for identification of TRPML3 blockers.
Example 20
[0551] As shown in the experiment contained in FIG. 20, expression
of wild-type TRPML3 yields low sodium current levels,
codon-optimized wild-type TRPML3 yields intermediate sodium current
levels, and A419P TRPML3 yields high sodium current levels. Thus,
codon-optimized wild-type TRPML3 and A419P TRPML3 facilitate
screening for compounds that modulate TRPML3 function.
Example 21
[0552] Screening oocytes injected with codon-optimized human TRPML3
cRNA
[0553] FIG. 21 contains an experiment involving screening oocytes
injected with codon-optimized human TRPML3 cRNA to identify a
compound (TRPML3 enhancer) that activates TRPML3. The results
contained therein illustrate the identification of an enhancer that
activates TRPML3 when oocytes are voltage clamped to -60 mV
[0554] In multiple oocytes, the TRPML3 enhancer increased TRPML3
activity by 169+/-26% from (representative trace on top) and had no
effect on uninjected oocytes with no TRPML3 expression
(representative trace on bottom). Addition of buffer only had no
effect on TRPML3 currents and the effects of the TRPML3 enhancer
were reproducible upon a second application.
Example 22
[0555] TRPML3 enhancer effect on TRPML3 I/V curve
[0556] FIG. 22 contains an experiment illustrating an example of
TRPML3 enhancer effect on TRPML3 I/V curve. This figure shows that
the same enhancer as in prior example activates TRPML3 at negative
voltages in an I/V curve analysis. Oocytes injected with
codon-optimized human TRPML3 cRNA were untreated (blue triangles
labeled control) or stimulated with TRPML3 enhancer (magenta
squares labeled enhancer) and currents were measured at voltages
from -90 to +30 mV. Note that the TRPML3 enhancer activates TRPML3
current at negative voltages (inward currents are larger with
enhancer compared to with control), resulting in an increase in the
slope of the I/V curve. Note also that the zero current shifts to
the right, indicating an increased sodium conductance in the
presence of the enhancer.
Example 23
[0557] FIG. 23 contains an experiment which exemplifies TRPML3
enhancer effect in the presence and absence of extracellular
sodium. Oocytes expressing codon-optimized human TRPML3 cRNA were
stimulated with NMDG (no sodium), TRPML3 enhancer plus sodium,
buffer only, or TRPML3 enhancer plus NMDG (no sodium). Note that
TRPML3 enhancer increased TRPML3 activity in the presence of sodium
but had no effect in the absence of sodium. These data demonstrate
that the TRPML3 enhancer opens TRPML3 channels and increases the
flow of sodium ions into the oocyte. FIG. 23 illustrates that this
same enhancer does not activate TRPML3 in the absence of
extracellular sodium. Since this compound opens TRPML3 and
increases sodium flux into the cell, it is a candidate salty taste
enhancer. Collectively, these data demonstrate how the
electrophysiology oocyte assay can be used to identify TRPML3
enhancers that are candidate human salty taste enhancers, and how
wild-type codon-optimized TRPML3 facilitates identification of
TRPML3 enhancers in the oocyte electrophysiology assay.
Example 24
Expression level of WT TRPML3 in different mammalian cell types
[0558] As shown in the experiment contained in FIG. 24, the
expression level of WT TRPML3 depends on the mammalian cell type.
In panel A of the Figure is a current voltage analysis (I/V plot)
of HEK293 cells expressing WT and the A419P mutant TRPML3 channel.
It shows that the A419P mutant TRPML3 channels express large inward
rectifying currents (pink), whereas only small WT TRPML3 currents
are observed (blue). B. WT and A419P mutant TRPML3 channels have
similar functional characteristics in CHO cells.
Example 25
[0559] Use of TRPML3 for enhancer and blocker screening in CHO
cells
[0560] The experiment in FIG. 25 relates to the use of TRPML3 for
enhancer and blocker screening in CHO cells. Panel A shows WT human
TRPML3 channels transiently expressed in CHO cells which were used
to identify channel enhancers. I/V plot shows that compared to
buffer alone (blue; control), use of the enhancer results in an
increase in inward current at negative potentials (pink). Panel B.
shows mutant A419P TRPML3 channel stably expressed in CHO cells are
used to identify channel blockers. Compared to buffer alone (blue;
control) addition of 1 mM GdCl.sub.3 results in a decrease in
inward current (pink).
Example 26
[0561] Screening Assays Using Codon-Optimized TRPML3 in Mammalian
Cells
[0562] The experiment in FIG. 26 contains an experiment in
mammalian cells using the same codon optimized WT TRPML3 contained
in FIG. 19 for the screening of compounds which enhance TRPML3
function. Panel A shows the transient expression of non codon
optimized WT TRPML3 (light blue) results in little current in
HEK293 cells. In contrast, use of codon optimized WT TRPML3 (Dark
Blue; Cod Opt WT) results in currents with similar average
amplitude as A419P mutant channel (pink). Panel B shows the use of
codon optimized WT TRPML3 (blue) delivered with Baculovirus
transduction results in similar average currents as A419P TRPML3
(pink). Panel C. shows cells transduced with codon optimized WT
TRPML3 baculovirus is used to identify enhancers of TRPML3
function. Compared to buffer alone (blue; control) addition of
enhancer compound results in an increase in inward current
(pink).
Example 27
[0563] Coexpression of WT and A419P TRPML3
[0564] The experiment in FIG. 27 relates to an experiment involving
the coexpression of WT and A419P TRPML3. The results in the figure
indicate that this increases functional surface expression in
HEK293 cells. Panel A shows currents elicited from A419P TRPML3
cDNA (0.5 ug) transfected into HEK293 cells, yielding currents with
characteristic inward rectification. Panel B shows WT non codon
optimized TRPML3 (1.5 ug) is expressed in HEK293 cells and yields
no currents. Panel C shows the coexpression of A419P (0.5 ug) with
WT (1.5 ug) TRPML3 cDNAs in HEK293 cells result in large inward
currents which are twice as large as those when expressing A419P
cDNA alone. Panel D contains I/V plot of the average currents
elicited from WT (blue), A419P (pink) and coexpression of WT and
A419P (yellow) TRPML3 cDNAs in HEK293 cells.
Example 28
[0565] The experiment in FIG. 28 is an example of TRPML3 function
measured in an IonWorks PPC patch plate experiment. Panel A
contains a view of all 384 wells from a PPC patch plate with an
A419 TRPML3 inducible clone showing the results of the pre-compound
scan. Yellow indicates wells where the current at -120 mV was
.ltoreq.0 nA (in control experiments with parental CHO-K1 cells
none of the wells were labeled yellow). Blue indicates wells were
the average seal resistance was too low (<10 mOhm) to measure
the current reliably. A419P TRPML3 currents could be measured in
94% of the wells.
[0566] Panel B contains the average currents .+-.SEM before and
after addition of 4 mM GdCl3 or extracellular buffer (mock
addition) from the patch plate shown in A. GdCl3 was added to
column 1-38 while extracellular buffer was added to columns 39-48.
For comparison, data is included from a separate experiment with
parental CHO-K1 cells. The stability of the TRPML3 current after
mock addition indicates that the assay should detect compounds that
either enhance or block TRPML3 currents.
Example 29
[0567] FIG. 29 contains an example of an IonWorks scan with an
inducible CHO-K1 cell line expressing A419P TRPML3 (top panel).
TRPML3 inwardly rectifies, denoted by more current at
hyperpolarized potentials (more negative potentials) and less
current at depolarized potentials (more positive potentials).
Addition of GdCl3 blocks TRPML3 current. Red line denotes scan in
sodium (NaCl) solution. Blue line denotes scan in 4 mM GdCl3
solution. The middle panel is from parental CHO-K1 cells used as a
negative control. The positive currents at negative potentials are
due to leak subtraction overcorrecting the current at negative
potentials. The bottom panel show the voltage command protocol used
to record currents. The step from 0 mV to 10 mV is used to
calculate the leak current (current flowing through leaks in the
seal) which is subtracted from the total current to obtain the
current flowing through the membrane.
[0568] Results are from single wells in a PPC patch plate and
represent the average current of up to 64 cells. FIG. 30 contains a
flowchart of experiments used to examine the effect of compounds on
human TRPML3 (hTRPML3) activity in the IonWorks assay.
Example 30
[0569] As shown by the results of the real-time PCR experiment
contained in FIG. 31, TRPML3 taste cells are specifically ablated
from taste buds in the Varitint waddler mice. End-point RT-PCR
experiments on taste buds (TB) and lingual epithelial cells (LE) of
Varitint waddler (Va) or wild-type (WT) mice isolated by
laser-capture microdissection are shown.
[0570] The results indicate that TRPML3 is only expressed in taste
buds of WT mice and absent in taste buds of Va mice, whereas all
other taste genes (T1R2, Gpr113, TRPM5) as well as housekeeping
genes (beta-actin, GAPDH) are equally expressed in TB and LE. `+`
indicates that reverse transcription was performed and `-`
indicates that no reverse transcription was performed. PCR bands
were only observed with reverse transcriptase indicating that PCR
products are derived from mRNA and not genomic DNA.
Example 31
[0571] This example relates to experiments summarized in FIG. 32
containing the results of a teal-time PCR experiment showing that
TRPML3 cells are specifically ablated from taste buds of Varitint
waddler mice. Real-time quantitative RT-PCR experiments on taste
buds of Varitint waddler (Va) or wild-type (WT) mice isolated by
laser-capture microdissection. TRPML3 is only expressed in taste
buds of WT mice and absent in taste buds of Va mice (similar
results were obtained using two different primer sets labeled
Mcoln3.sub.--1 and Mcoln3.sub.--2), whereas all other taste genes
(Tas1r2, Tas1r3, PKD2l1, TRPM5, Plcb2, Tas2r108, and Tas2r116) as
well as a housekeeping gene (control) are expressed in taste buds
from Va and WT mice.
Example 32
[0572] This example relates to an experiment in FIG. 33 showing
that sweet, bitter, umami and sour taste cells remain intact in the
Varitint waddler mice. In situ hybridization of circumvallate
papilla from the back of the tongue of wild-type (top row of
images) and Varitint waddler (Va; bottom row of images) mice.
PKD1L3 (left; sour), PKD2L1 (middle; sour), and TRPM5 (right;
sweet, bitter, and umami) taste cells were present at similar
levels in wild-type and Va mice.
Example 33
[0573] This example relates to an experiment using CT nerve
recordings in wild-type and Varitint waddler mice stimulated with
salty taste stimuli contained in FIG. 34 showing that the Varitint
waddler mice are deficient in salty taste perception and that the
wild-type mice detected salty taste under the same conditions. CT
nerve recordings from wild-type (left) or Varitint waddler (Va;
right) mice. Anterior tongues were stimulated with 0.1 M NaCl or
0.1 M NaCl plus 5 uM benzamil to inhibit the amiloride-sensitive
component of the CT nerve response. Tongues were rinsed with a low
salt solution containing 10 mM KCl in between NaCl stimulations.
Note that the benzamil-insensitive component of the CT nerve
response is largely eliminated in the Va mouse (red arrows),
indicating that ablation of TRPML3 taste cells significantly
impairs salty taste perception. In addition, the immediate phasic
response to NaCl is greatly reduced in the Va mouse (red circles).
Scale bars indicate time frames of salt application (x-axis) and
the magnitude of the CT response (y-axis; arbitrary units).
[0574] FIG. 35 contains an alignment of the protein sequences
derived from human (NM.sub.--018298) and mouse (NM.sub.--134160)
TRPML3 sequences wherein human is denoted Hs and mouse is denoted
Mm. Human and mouse protein sequences are 91% identical and 96%
similar. The six transmembrane domains in the TRPML3 polypeptides
for both human and mouse TRPML3 are underlined TM1 through TM6. The
pore region between TM5 and TM6 is denoted `pore region`. The amino
and carboxy termini are predicted to be located inside the cell.
The A419P mutation discussed herein and found in the
varitint-waddler mouse locks TRPML3 in the open conformation is in
TM5 and is highlighted in red. Another mutation, V412P, partially
activates TRPML3 and is denoted in magenta.
[0575] While the invention has been described by way of examples
and preferred embodiments, it is understood that the words which
have been used herein are words of description, rather than words
of limitation. Changes may be made, within the purview of the
appended claims, without departing from the scope and spirit of the
invention in its broader aspects. Although the invention has been
described herein with reference to particular means, materials, and
embodiments, it is understood that the invention is not limited to
the particulars disclosed. The invention extends to all equivalent
structures, means, and uses which are within the scope of the
appended claims.
[0576] As afore mentioned the taste cell specific genes identified
according to the invention and the corresponding gene products and
cells which express same e.g., endogenous taste or chemosensory
cells and recombinant cells including these taste specific genes,
and their orthologs, allelic variants, variants possessing at least
90% sequence identity thereto and/or genes which specifically
hybridize thereto under hybridization conditions denied supra may
be used in assays to identify taste modulatory compounds as well as
in therapeutic screening assays.
[0577] For example these taste specific genes, polypeptides and
cells expressing same can be used to screen for compounds for
treatment of digestive system disorders. These disorders include by
way of example conditions affecting digestion such as dyspepsia,
autoimmune and inflammatory diseases affecting the digestive system
such as ulcerative colitis, inflammatory bowel syndrome, Crohn's
syndrome, celiac disease, and precancers and cancers that affect
the digestive system such as cancers affecting the salivary glands,
taste buds, stomach, pancreas, gall bladder, esophagus, small or
large intestine, anus or colon.
[0578] Also these taste specific genes may be used in screening
assays to identify compounds that affect taste cell turnover. It is
known that taste cells turnover rapidly (about every couple of
weeks). Moreover, many conditions including chemotherapy or
radiation treatment, as well as old age may negatively affect the
ability of taste cells to develop. The result is a diminished sense
of taste which may result in a decreased quality of life in cancer
patients or the elderly. This is particularly pronounced in
patients with head and neck cancer, esophageal, stomach, lung, or
pancreatic cancers. Additionally, this may evolve along with
another condition, cachexia or wasting syndrome that combines to
reduce the desire to eat. Lack of proper nutrition is a serious
cause of morbidity and mortality in cancer patients. The subject
taste specific genes contain genes expressed in stem cells
suggesting that they are markers of stem cells that are the
precursors of and which evolve into taste cells. These genes or
cells which express same can be used to identify signals that
accelerate taste cell development. These signals which likely
comprise cytokine-like receptors present on taste cells likely
mediate taste cell development and can be used in screens to
identify compounds that induce taste cell differentiation or
proliferation. The ligands therefore potentially may be isolated
from saliva and may account for the ability of saliva to influence
taste function. For example, patients with Sjogren's syndrome (an
autoimmune disease that attacks the salivary glands) exhibit
altered taste functions. The subject genes and the study of gene
expression in the salivary glands by use of gene arrays will
facilitate an understanding of these differentiation
mechanisms.
[0579] The subject taste cell specific genes and corresponding gene
products and cells which express these genes may also be used in
order to identify potential therapeutics for modulating the immune
system of the oral cavity. The oral cavity is populated by normal
flora as is the digestive tract. Alterations in normal flora may
give rise to conditions such as gingivitis, halitosis, gastric
problems and other infections that may result in tooth decay or
tooth loss. Included within the taste cell specific genes
identified herein are a number of immune system genes. These genes
and the corresponding polypeptides or cells which express same can
be used to identify therapeutics for maintaining immune homeostasis
in the oral cavity, preventing overgrowth of pathogenic microbia,
and for identification of the cell types in the oral cavity that
are the key players in maintaining proper oral cavity immunity.
[0580] Moreover, the subject taste cell specific genes and the
corresponding gene products or cells which express same are useful
in screening assays for identifying compounds for treatment of
diabetes, eating disorders such as obesity.
[0581] Gene products and compounds that enhance or inhibit gene
products identified by the inventors can affect: oral hygiene,
halitosis, detoxification of noxious substances in anorexia,
bulimia, and other metabolic disorders. The expression of taste
receptors in the digestive system likely represents a comprehensive
system that detects food and different types at different places
during digestion. Therefore, "sensing" the presence of food or
specific types such as carbohydrates, fats, umami foods, salts,
should trigger various signals that may regulate the production of
molecules that participate in the regulation of digestion such as
GIP (glucose-dependent insulinotrophic polypeptide) and GLP-1
(glucagon-like peptide 1) produced by the enter endocrine cells in
the intestine. It is likely that taste receptors on these cells
regulate the production of other molecular signals in other cells
of the digestive system when triggered. These phenomena may be
studied by determining which cells express different receptors and
then using gene arrays to study the molecules that these cells
produce when activated.
[0582] More specifically, this invention identifies and provides
functional (electrophysiological) and immohistochemistry data which
indicate that TRPML3 (MCOLN3) encodes a polypeptide that functions
as a primate (e.g., human) salty taste receptor
[0583] Also more specifically, the present invention provides the
use of these taste specific genes as markers which can be used to
enrich, identify or isolate salt receptor expressing cells.
[0584] Also more specifically, this invention provides in vitro and
in vivo assays which use TRPML3 (MCOLN3) and TRPML3 expressing
cells or TRPML3 transgenic animal models to identify agonist,
antagonist or enhancer compounds which elicit or modulate (block or
enhance) salty taste in primates including humans. These assays use
cells which express TRPML3 alone or cells which express the TRPML3
ion channel in association with other taste specific polypeptides
such as NALCN or NKAIN3.
[0585] Also more specifically, this invention provides transgenic
animals, preferably rodents, and the use thereof to confirm the
role of TRPML3 in salty taste in mammals and in other physiological
functions involving sodium and other ions such as sodium
metabolism, blood pressure, fluid retention and excretion, urinary
function and cardiac function.
[0586] Also more specifically, this invention provides in vitro and
in vivo assays which use TRPML3 and TRPML3 expressing cells or
transgenic animals in assays, preferably electrophysiological
assays in order to identify therapeutic compounds which alleviate
diseases and conditions involving deficiencies in the expression of
this polypeptide including hyperexpression, hyporexpression, and
mutations in the TRPML3 polypeptide that affect its ability to
function as a taste specific sodium channel in a mammal including
e.g., human and non-human primates and rodents. These conditions
include by way of example Addison's disease and diseases involving
aberrant aldosterone production or vasopressin release such as
hypertension, hypotension, fluid retention, and impaired urinary or
cardiac function such as arrhythmia, heart attach and stroke. In
addition conditions treatable using TRPML3 modulatory compounds
include melanoma and other conditions involving melanocytes such as
pigmentation disorders.
[0587] Therefore, in conclusion this invention relates to the
identification of MCOLN3 as encoding a human salty taste receptor
which allows for the design of screening assays using cells
transfected with this gene for the purpose of identifying agonists,
antagonists or enhancers (modulator compounds) which affect the
function of this molecule. These compounds can be used as taste
modulators and as therapeutics that modulate sodium metabolism,
absorption and excretion. In order to further describe the
invention and exemplary embodiments, the following TRPML3 nucleic
acid and polypeptide sequences from different mammals including
human are provided below. However, as afore-mentioned, the taste
cell specific genes identified according to the invention and the
corresponding gene products and cells which express same e.g.,
endogenous taste or chemosensory cells and recombinant cells
including these taste specific genes, and their orthologs, allelic
variants, chimeras, and genetically engineered fragments and
variants possessing at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or
greater sequence identity thereto and/or genes which specifically
hybridize thereto under hybridization conditions denied supra may
be used in assays to identify taste modulatory compounds as well as
in therapeutic screening assays.
TABLE-US-00002 SEQUENCE LISTING (EXEMPLARY TRPML3, TRPML2, TRPML1
POLYPEPTIDE AND NUCLEIC ACID SEQUENCES AND NKAIN3 AND NALCN
SEQUENCES) Homo sapiens TRPML3 (NM_018298) (SEQ ID NO:1)
ATGGCAGATCCTGAGGTAGTTGTGAGTAGCTGCAGCTCTCATGAAGAGGAAAATCGCTGCAATTTTAACC
AGCAAACATCTCCATCTGAGGAGCTTCTATTAGAAGACCAGATGAGGCGAAAACTCAAATTTTTTTTCAT
GAATCCCTGTGAGAAGTTCTGGGCTCGAGGTAGAAAACCATGGAAACTTGCCATACAAATTCTAAAAATT
GCAATGGTGACTATCCAGCTGGTCTTATTTGGGCTAAGTAACCAGATGGTGGTAGCTTTCAAGGAAGAGA
ATACTATAGCATTCAAACACCTTTTCCTAAAAGGATATATGGACCGAATGGATGACACATATGCAGTGTA
CACACAAAGTGACGTGTATGATCAGTTAATCTTCGCAGTAAACCAGTACTTGCAGCTATACAATGTCTCC
GTTGGGAATCATGCTTATGAGAACAAAGGTACCAAGCAATCTGCTATGGCAATCTGTCAGCACTTCTACA
AGCGAGGAAACATCTACCCTGGAAATGATACCTTTGACATCGATCCAGAAATTGAAACTGAGTGTTTCTT
TGTGGAGCCAGATGAACCTTTTCACATTGGGACACCAGCAGAAAATAAACTGAACTTAACACTGGACTTC
CACAGACTCCTAACAGTGGAGCTTCAGTTTAAACTGAAAGCCATTAATCTGCAGACAGTTCGTCATCAAG
AACTCCCTGACTGTTATGACTTTACTCTGACTATAACATTTGACAACAAGGCCCATAGTGGAAGAATTAA
AATAAGTTTAGATAATGACATTTCCATCAGAGAATGTAAAGACTGGCATGTATCTGGATCAATTCAGAAG
AACACTCATTACATGATGATCTTTGATGCCTTTGTCATTCTGACTTGCTTGGTTTCATTAATCCTCTGCA
TTAGATCTGTGATTAGAGGACTTCAGCTTCAGCAGGAGTTTGTCAATTTTTTCCTCCTCCATTATAAGAA
GGAAGTTTCTGTTTCTGATCAAATGGAATTTGTCAATGGATGGTACATTATGATTATTATTAGTGACATA
TTGACAATCATTGGATCAATTCTAAAAATGGAAATCCAAGCTAAGAGTCTAACTAGTTATGATGTCTGTA
GCATACTTCTTGGGACTTCTACCATGCTCGTGTGGCTTGGAGTCATCCGATACCTCGGTTTCTTTGCAAA
GTACAACCTCCTCATTTTGACCCTTCAGGCAGCGCTGCCCAATGTCATCAGGTTCTGCTGCTGTGCAGCT
ATGATTTACTTAGGTTACTGCTTCTGTGGATGGATCGTGCTGGGGCCTTACCATGACAAGTTTCGTTCTC
TGAACATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGATATGTTTGCCACGTTTGCAAAAAT
GCAGCAAAAAAGTTACTTAGTCTGGCTGTTTAGTAGAATTTACCTCTACTCATTCATCAGCCTCTTTATA
TATATGATTTTAAGTCTTTTCATTGCACTGATCACTGATACATACGAAACAATTAAGCAATACCAACAAG
ATGGCTTCCCAGAGACTGAACTTCGTACATTTATATCAGAATGCAAAGATCTACCCAACTCTGGAAAATA
CAGATTAGAAGATGACCCTCCAGTATCTTTATTCTGCTGTTGTAAAAAG (SEQ ID NO:1)
Homo sapiens TRPML3 (NM_018298) (SEQ ID NO:2)
MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKI
AMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVS
VGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDF
HRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQK
NTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKEVSVSDQMEFVNGWYIMIIISDI
LTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCAA
MIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFI
YMILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVSLFCCCKK
(SEQ ID NO:2) Homo sapiens TRPML3 A419P (SEQ ID NO:3)
ATGGCAGATCCTGAGGTAGTTGTGAGTAGCTGCAGCTCTCATGAAGAGGAAAATCGCTGCAATTTTAACC
AGCAAACATCTCCATCTGAGGAGCTTCTATTAGAAGACCAGATGAGGCGAAAACTCAAATTTTTTTTCAT
GAATCCCTGTGAGAAGTTCTGGGCTCGAGGTAGAAAACCATGGAAACTTGCCATACAAATTCTAAAAATT
GCAATGGTGACTATCCAGCTGGTCTTATTTGGGCTAAGTAACCAGATGGTGGTAGCTTTCAAGGAAGAGA
ATACTATAGCATTCAAACACCTTTTCCTAAAAGGATATATGGACCGAATGGATGACACATATGCAGTGTA
CACACAAAGTGACGTGTATGATCAGTTAATCTTCGCAGTAAACCAGTACTTGCAGCTATACAATGTCTCC
GTTGGGAATCATGCTTATGAGAACAAAGGTACCAAGCAATCTGCTATGGCAATCTGTCAGCACTTCTACA
AGCGAGGAAACATCTACCCTGGAAATGATACCTTTGACATCGATCCAGAAATTGAAACTGAGTGTTTCTT
TGTGGAGCCAGATGAACCTTTTCACATTGGGACACCAGCAGAAAATAAACTGAACTTAACACTGGACTTC
CACAGACTCCTAACAGTGGAGCTTCAGTTTAAACTGAAAGCCATTAATCTGCAGACAGTTCGTCATCAAG
AACTCCCTGACTGTTATGACTTTACTCTGACTATAACATTTGACAACAAGGCCCATAGTGGAAGAATTAA
AATAAGTTTAGATAATGACATTTCCATCAGAGAATGTAAAGACTGGCATGTATCTGGATCAATTCAGAAG
AACACTCATTACATGATGATCTTTGATGCCTTTGTCATTCTGACTTGCTTGGTTTCATTAATCCTCTGCA
TTAGATCTGTGATTAGAGGACTTCAGCTTCAGCAGGAGTTTGTCAATTTTTTCCTCCTCCATTATAAGAA
GGAAGTTTCTGTTTCTGATCAAATGGAATTTGTCAATGGATGGTACATTATGATTATTATTAGTGACATA
TTGACAATCATTGGATCAATTCTAAAAATGGAAATCCAAGCTAAGAGTCTAACTAGTTATGATGTCTGTA
GCATACTTCTTGGGACTTCTACCATGCTCGTGTGGCTTGGAGTCATCCGATACCTCGGTTTCTTTGCAAA
GTACAACCTCCTCATTTTGACCCTTCAGGCAGCGCTGCCCAATGTCATCAGGTTCTGCTGCTGTCCAGCT
ATGATTTACTTAGGTTACTGCTTCTGTGGATGGATCGTGCTGGGGCCTTACCATGACAAGTTTCGTTCTC
TGAACATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGATATGTTTGCCACGTTTGCAAAAAT
GCAGCAAAAAAGTTACTTAGTCTGGCTGTTTAGTAGAATTTACCTCTACTCATTCATCAGCCTCTTTATA
TATATGATTTTAAGTCTTTTCATTGCACTGATCACTGATACATACGAAACAATTAAGCAATACCAACAAG
ATGGCTTCCCAGAGACTGAACTTCGTACATTTATATCAGAATGCAAAGATCTACCCAACTCTGGAAAATA
CAGATTAGAAGATGACCCTCCAGTATCTTTATTCTGCTGTTGTAAAAAG (SEQ ID NO:3)
Homo sapiens TRPML3 A419P (SEQ ID NO:4)
MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKI
AMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVS
VGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDF
HRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQK
NTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKEVSVSDQMEFVNGWYIMIIISDI
LTIIGSILIKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCP
AMIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYSFISLFIY
MILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVSLFCCCKK (SEQ
ID NO:4) Homo sapiens TRPML1 (NM_020533) (SEQ ID NO:5)
ATGACAGCCCCGGCGGGTCCGCGCGGCTCAGAGACCGAGCGGCTTCTGACCCCCAACCCCGGGTATGGGA
CCCAGGCGGGGCCTTCACCGGCCCCTCCGACACCCCCAGAAGAGGAAGACCTTCGCCGTCGTCTCAAATA
CTTTTTCATGAGTCCCTGCGACAAGTTTCGAGCCAAGGGCCGCAAGCCCTGCAAGCTGATGCTGCAAGTG
GTCAAGATCCTGGTGGTCACGGTGCAGCTCATCCTGTTTGGGCTCAGTAATCAGCTGGCTGTGACATTCC
GGGAAGAGAACACCATCGCCTTCCGACACCTCTTCCTGCTGGGCTACTCGGACGGAGCGGATGACACCTT
CGCAGCCTACACGCGGGAGCAGCTGTACCAGGCCATCTTCCATGCTGTGGACCAGTACCTGGCGTTGCCT
GACGTGTCACTGGGCCGGTATGCGTATGTCCGTGGTGGGGGTGACCCTTGGACCAATGGCTCAGGGCTTG
CTCTCTGCCAGCGGTACTACCACCGAGGCCACGTGGACCCGGCCAACGACACATTTGACATTGATCCGAT
GGTGGTTACTGACTGCATCCAGGTGGATCCCCCCGAGCGGCCCCCTCCGCCCCCCAGCGACGATCTCACC
CTCTTGGAAAGCAGCTCCAGTTACAAGAACCTCACGCTCAAATTCCACAAGCTGGTCAATGTCACCATCC
ACTTCCGGCTGAAGACCATTAACCTCCAGAGCCTCATCAATAATGAGATCCCGGACTGCTATACCTTCAG
CGTCCTGATCACGTTTGACAACAAAGCACACAGTGGGCGGATCCCCATCAGCCTGGAGACCCAGGCCCAC
ATCCAGGAGTGTAAGCACCCCAGTGTCTTCCAGCACGGAGACAACAGCTTCCGGCTCCTGTTTGACGTGG
TGGTCATCCTCACCTGCTCCCTGTCCTTCCTCCTCTGCGCCCGCTCACTCCTTCGAGGCTTCCTGCTGCA
GAACGAGTTTGTGGGGTTCATGTGGCGGCAGCGGGGACGGGTCATCAGCCTGTGGGAGCGGCTGGAATTT
GTCAATGGCTGGTACATCCTGCTCGTCACCAGCGATGTGCTCACCATCTCGGGCACCATCATGAAGATCG
GCATCGAGGCCAAGAACTTGGCGAGCTACGACGTCTGCAGCATCCTCCTGGGCACCTCGACGCTGCTGGT
GTGGGTGGGCGTGATCCGCTACCTGACCTTCTTCCACAACTACAATATCCTCATCGCCACACTGCGGGTG
GCCCTGCCCAGCGTCATGCGCTTCTGCTGCTGCGTGGCTGTCATCTACCTGGGCTACTGCTTCTGTGGCT
GGATCGTGCTGGGGCCCTATCATGTGAAGTTCCGCTCACTCTCCATGGTGTCTGAGTGCCTGTTCTCGCT
CATCAATGGGGACGACATGTTTGTGACGTTCGCCGCCATGCAGGCGCAGCAGGGCCGCAGCAGCCTGGTG
TGGCTCTTCTCCCAGCTCTACCTTTACTCCTTCATCAGCCTCTTCATCTACATGGTGCTCAGCCTCTTCA
TCGCGCTCATCACCGGCGCCTACGACACCATCAAGCATCCCGGCGGCGCAGGCGCAGAGGAGAGCGAGCT
GCAGGCCTACATCGCACAGTGCCAGGACAGCCCCACCTCCGGCAAGTTCCGCCGCGGGAGCGGCTCGGCC
TGCAGCCTTCTCTGCTGCTGCGGAAGGGACCCCTCGGAGGAGCATTCGCTGCTGGTGAAT (SEQ
ID NO:5) Homo sapiens TRPML1 (NM_020533) (SEQ ID NO:6)
MTAPAGPRGSETERLLTPNPGYGTQAGPSPAPPTPPEEEDLRRRLKYFFMSPCDKFRAKGRKPCKLMLQV
VKILVVTVQLILFGLSNQLAVTFREENTIAFRHLFLLGYSDGADDTFAAYTREQLYQAIFHAVDQYLALP
DVSLGRYAYVRGGGDPWTNGSGLALCQRYYHRGHVDPANDTFDIDPMVVTDCIQVDPPERPPPPPSDDLT
LLESSSSYKNLTLKFHKLVNVTIHFRLKTINLQSLINNEIPDCYTFSVLITFDNKAHSGRIPISLETQAH
IQECKHPSVFQHGDNSFRLLFDVVVILTCSLSFLLCARSLLRGFLLQNEFVGFMWRQRGRVISLWERLEF
VNGWYILLVTSDVLTISGTIMKIGIEAKNLASYDVCSILLGTSTLLVWVGVIRYLTFFHNYNILIATLRV
ALPSVMRFCCCVAVIYLGYCFCGWIVLGPYHVKFRSLSMVSECLFSLINGDDMFVTFAAMQAQQGRSSLV
WLFSQLYLYSFISLFIYMVLSLFIALITGAYDTIKHPGGAGAEESELQAYIAQCQDSPTSGKFRRGSGSA
CSLLCCCGRDPSEEHSLLVN (SEQ ID NO:6) Homo sapiens TRPML2 (NM_153259)
(SEQ ID NO:7)
ATGGCCCGGCAGCCTTATCGTTTTCCCCAGGCAAGGATTCCGGAGAGAGGATCAGGTGTTTTCAGGTTAA
CCGTCAGAAACGCAATGGCACATCGTGATTCTGAGATGAAAGAAGAATGTCTAAGGGAAGACCTGAAGTT
TTACTTCATGAGCCCTTGTGAAAAATACCGAGCCAGACGCCAGATTCCGTGGAAACTGGGTTTGCAGATT
TTGAAGATAGTCATGGTCACCACACAGCTTGTTCGTTTTGGTTTAAGTAACCAGCTGGTGGTTGCTTTCA
AAGAAGATAACACTGTTGCTTTTAAGCACTTGTTTTTGAAAGGATATTCTGGTACAGATGAAGATGACTA
CAGCTGCAGTGTATATACTCAAGAGGATGCCTATGAGAGCATCTTTTTTGCTATTAATCAGTATCATCAG
CTAAAGGACATTACCCTGGGGACCCTTGGTTATGGAGAAAATGAAGACAATAGAATTGGCTTAAAAGTCT
GTAAGCAGCATTACAAGAAAGGGACCATGTTTCCTTCTAATGAGACACTGAATATTGACAACGACGTTGA
GCTCGATTGTGTTCAATTAGACCTTCAGGACCTCTCCAAGAAGCCTCCGGACTGGAAGAACTCATCATTC
TTCAGACTGGAATTTTATCGGCTCTTACAGGTTGAAATCTCCTTTCATCTTAAAGGCATTGACCTACAGA
CAATTCATTCCCGTGAGTTACCAGACTGTTATGTCTTTCAGAATACGATTATCTTTGACAATAAAGCTCA
CAGTGGCAAAATCAAAATCTATTTTGACAGTGATGCCAAAATTGAAGAATGTAAAGACTTGAACATATTT
GGATCTACTCAGAAAAATGCTCAGTATGTCCTGGTGTTTGATGCATTTGTCATTGTGATTTGCTTGGCAT
CTCTTATTCTGTGTACAAGATCCATTGTTCTTGCTCTAAGGTTACGGAAGAGATTTCTAAATTTCTTCCT
GGAGAAGTACAAGCGGCCTGTGTGTGACACCGACCAGTGGGAGTTCATCAACGGCTGGTATGTCCTGGTG
ATTATCAGCGACCTAATGACAATCATTGGCTCCATATTAAAAATGGAAATCAAAGCAAAGAATCTCACAA
ACTATGATCTCTGCAGCATTTTTCTTGGAACCTCTACGCTCTTGGTTTGGGTTGGAGTCATCAGATACCT
GGGTTATTTCCAGGCATATAATGTGCTGATTTTAACAATGCAGGCCTCACTGCCAAAAGTTCTTCGGTTT
TGTGCTTGTGCTGGTATGATTTATCTGGGTTACACATTCTGTGGCTGGATTGTCTTAGGACCATACCATG
ACAAGTTTGAAAATCTGAACACAGTTGCTGAGTGTCTGTTTTCTCTGGTCAACGGTGATGACATGTTTGC
AACCTTTGCCCAAATCCAGCAGAAGAGCATCTTGGTGTGGCTGTTCAGTCGTCTGTATTTATATTCCTTC
ATCAGCCTTTTTATATATATGATTCTCAGTCTTTTTATTGCACTTATTACAGATTCTTATGACACCATTA
AGAAATTCCAACAGAATGGGTTTCCTGAAACGGATTTGCAGGAATTCCTGAAGGAATGCAGTAGCAAAGA
AGAGTATCAGAAAGAGTCCTCAGCCTTCCTGTCCTGCATCTGCTGTCGGAGGAGGAAAAGAAGTGATGAT
CACTTGATACCTATTAGC (SEQ ID NO:7) Homo sapiens TRPML2 (NM_153259)
(SEQ ID NO:8)
MARQPYRFPQARIPERGSGVFRLTVRNAMAHRDSEMKEECLREDLKFYFMSPCEKYRARRQIPWKLGLQI
LKIVMVTTQLVRFGLSNQLVVAFKEDNTVAFKHLFLKGYSGTDEDDYSCSVYTQEDAYESIFFAINQYHQ
LKDITLGTLGYGENEDNRIGLKVCKQHYKKGTMFPSNETLNIDNDVELDCVQLDLQDLSKKPPDWKNSSF
FRLEFYRLLQVEISFHLKGIDLQTIHSRELPDCYVFQNTIIFDNKAHSGKIKIYFDSDAKIEECKDLNIF
GSTQKNAQYVLVFDAFVIVICLASLILCTRSIVLALRLRKRFLNFFLEKYKRPVCDTDQWEFINGWYVLV
IISDLMTIIGSILKMEIKAKNLTNYDLCSIFLGTSTLLVWVGVIRYLGYFQAYNVLILTMQASLPKVLRF
CACAGMIYLGYTFCGWIVLGPYHDKFENLNTVAECLFSLVNGDDMFATFAQIQQKSILVWLFSRLYLYSF
ISLFIYMILSLFIALITDSYDTIKKFQQNGFPETDLQEFLKECSSKEEYQKESSAFLSCICCRRRKRSDD
HLIPIS (SEQ ID NO:8) Mus musculus TRPML3 (NM_134160) (SEQ ID NO:9)
ATGGCAAATCCCGAGGTGCTGGTTAGCAGCTGCAGAGCTCGCCAAGATGAAAGCCCCTGCACTTTCCACC
CGAGCTCGTCCCCGTCAGAGCAGCTTCTCTTAGAAGACCAGATGAGGCGGAAACTCAAGTTCTTTTTTAT
GAATCCTTGTGAGAAGTTCTGGGCTCGGGGTAGGAAGCCATGGAAACTTGCCATACAGATTCTGAAAATC
GCGATGGTGACTATCCAGCTGGTTCTGTTTGGACTAAGTAACCAGATGGTAGTAGCTTTCAAAGAGGAGA
ACACTATAGCCTTCAAACACCTCTTCCTAAAGGGCTACATGGATCGAATGGACGACACCTATGCAGTGTA
CACTCAGAGTGAAGTGTATGACCAGATCATCTTTGCAGTGACCCAGTACTTGCAGCTTCAGAACATCTCC
GTGGGCAATCACGCTTATGAGAACAAGGGGACTAAGCAGTCGGCGATGGCAATCTGTCAGCACTTCTACA
GGCAAGGAACCATCTGCCCCGGGAACGACACCTTTGACATCGATCCAGAAGTTGAAACAGAATGTTTCCT
TGTAGAGCCAGATGAAGCTTCCCACCTTGGAACGCCTGGAGAAAATAAACTCAACCTGAGCCTGGACTTC
CACAGACTTCTGACGGTGGAGCTCCAGTTTAAGCTCAAAGCCATCAATCTGCAGACAGTTCGACACCAGG
AGCTTCCTGACTGTTACGACTTTACGCTGACTATAACATTCGACAACAAGGCTCACAGTGGAAGAATCAA
AATAAGCTTAGACAACGACATTTCTATCAAAGAATGCAAAGACTGGCATGTGTCTGGATCAATTCAGAAG
AACACACACTACATGATGATCTTTGATGCCTTTGTCATTCTGACCTGCTTGGCCTCACTGGTGCTGTGTG
CCAGGTCTGTGATTAGGGGTCTTCAGCTTCAGCAGGAGTTTGTCAACTTCTTCCTTCTTCACTACAAGAA
GGAAGTTTCGGCCTCTGATCAGATGGAGTTCATCAACGGGTGGTACATTATGATCATCATTAGTGACATA
TTGACAATCGTTGGATCAGTTCTGAAAATGGAAATCCAAGCCAAGAGTCTCACAAGCTATGATGTCTGCA
GCATACTTCTCGGGACGTCAACTATGCTCGTGTGGCTTGGAGTTATCCGATACCTGGGTTTCTTTGCGAA
GTACAATCTCCTTATTCTGACCCTCCAGGCAGCGCTGCCCAACGTCATGAGGTTCTGTTGCTGCGCTGCT
ATGATCTATCTAGGCTATTGCTTTTGCGGATGGATTGTGCTGGGCCCTTACCATGAGAAGTTCCGTTCCC
TGAACAGGGTCTCCGAGTGCCTGTTCTCGCTGATAAACGGAGACGATATGTTTTCCACATTTGCGAAAAT
GCAGCAGAAGAGTTACCTGGTGTGGCTGTTCAGCCGAGTCTACCTGTACTCGTTCATCAGCCTCTTCATT
TACATGATTCTGAGCCTTTTCATCGCGCTCATCACAGACACATACGAAACAATTAAGCACTACCAGCAAG
ATGGCTTCCCAGAGACGGAACTTCGAAAGTTTATAGCGGAATGCAAAGACCTCCCCAACTCCGGAAAATA
CAGATTAGAAGATGACCCTCCGGGTTCTTTACTCTGCTGCTGCAAAAAG (SEQ ID NO:9) Mus
musculus TRPML3 (NM_134160) (SEQ ID NO:10)
MANPEVLVSSCRARQDESPCTFHPSSSPSEQLLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKI
AMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSEVYDQIIFAVTQYLQLQNIS
VGNHAYENKGTKQSAMAICQHFYRQGTICPGNDTFDIDPEVETECFLVEPDEASHLGTPGENKLNLSLDF
HRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIKECKDWHVSGSIQK
NTHYMMIFDAFVILTCLASLVLCARSVIRGLQLQQEFVNFFLLHYKKEVSASDQMEFINGWYIMIIISDI
LTIVGSVLKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVMRFCCCAA
MIYLGYCFCGWIVLGPYHEKFRSLNRVSECLFSLINGDDMFSTFAKMQQKSYLVWLFSRVYLYSFISLFI
YMILSLFIALITDTYETIKHYQQDGFPETELRKFIAECKDLPNSGKYRLEDDPPGSLLCCCKK
(SEQ ID NO:10) Gallus gallus TRPML3 (XM_426647) (SEQ ID NO:11)
ATGATCACCCGTGGTTTCCGTTCAGGCTTTTACGGCTGTAGCAATCGATATAGTGGTGCTAAGTGCCAGT
TAGTTGCTCAGACGTTTGTCATTTCAGCAATGGAGACTCCTGAAGTGGCTGTAAGCAGCTGCAGTGCTCG
GGATGACGAAGGGCTCTGCAGCTACGGACAACACCTATTGCTGCCACAAGAGCTGGTGGCGGAAGACCAG
CTGAGGAGGAAGCTGAAGTTCTTCTTCATGAACCCATGTGAAAAATTCTGGGCTCGGGGCAGAAAACCTT
GGAAACTTGGGATTCAGCTGCTCAAAATAGCAATGGTTACCATTCAGCTGGTGCTTTTTGGATTGAGCAA
TCAAATGGTGGTTGCTTTCAAAGAAGAGAACACTATTGCATTCAAACATCTCTTCTTGAAAGGGTACATG
GACAGAATGGATGATACCTATGCGGTATACACACAGACAGATGTCTATGACCAAATATTCTTTGCCATCA
ATCAGTACTTACAGTTGCCCAACATTTCTGTTGGAAACCATGCTTATGAGAAGAAAGGAGCAGAAGAGAC
AGCTCTGGCTGTATGTCAACAGTTCTACAAGCAAGGAACCATCTGTCCTGGAAATGACACCTTTGATATA
GACCCAGAGATTGTGACTGACTGCTTGTACATTGAGCCGATGATGTCTTTAGACAACAGAACAGTGGGAA
AGCACAATTTGAATTTCACTCTGGATTTCCACAGGCTCGTGGCAGTGCAACTCATGTTCAATCTGAAGGC
AATCAACCTCCAGACCGTCCGTCACCACGAGCTCCCTGACTGTTACGATTTCACCCTGACGATAGTGTTT
GATAATAAAGCCCACAGTGGAAGAATCAAAATCAGTCTAGACAACGACATAGAGATCAGGGAATGTAAAG
ACTGGCACGTTTCTGGATCAATACAGAAGAATACGCATTACATGATGATCTTCGATGCTTTTGTCATACT
GATCTGTCTGAGCTCATTGATCCTTTGCACTCGATCAGTAGTCAAAGGAATTCGGCTCCAAAGAGAATTT
GTAAGTTTTTTCCTATATTATTACAAGAAAGAGGTATCTTACAATGATCAGATGGAATTTGTCAATGGCT
GGTATATCCTCATTATGGTTAGTGATGTCCTCACTATCGTTGGATCAACTCTCAAAATGGAGATACAGGC
CAAGAGTCTGACAAGTTACGACGTCTGTAGCATACTCTTAGGAACATCCACTATGCTGGTGTGGCTTGGA
GTCATTCGCTACCTCGGTTTCTTTCAGAAGTATAATCTTCTCATTCTAACGCTGCGAGCAGCACTACCCA
ACGTCATGAGGTTCTGCTGTTGTGCTGCTATGATCTATCTAGGTTATTGTTTCTGCGGATGGATTGTACT
GGGGCCATACCACGTGAAGTTCCGTTCTCTGAATGTGGTTTCTGAATGCCTCTTTTCATTGATAAATGGA
GATGACATGTTTGCCACTTTTGCAAAAATGCAGCAGAAAAGTTACTTGGTTTGGTTATTCAGTAGAATCT
ACCTCTACTCCTTCATCAGCCTGTTCATCTACATGGTGCTAAGTCTCTTCATTGCACTCATTACAGATAC
ATATGAAACTATCAAGCACTACCAACAAGATGGCTTTCCAGAGACAGAACTTCAGAGATTTATATCACAG
TGCAAAGACTTACCAAACTCTGGAAGGTACAGATTAGAAGAGGAAGGTTCTGTATCTCTCTTCTGTTGTT
GCAGTGGTCCTAGTGAACATATC (SEQ ID NO:11) Gallus gallus TRPLM3
(XM_426647) (SEQ ID NO:12)
MITRGFRSGFYGCSNRYSGAKCQLVAQTFVISAMETPEVAVSSCSARDDEGLCSYGQHLLLPQELVAEDQ
LRRKLKFFFMNPCEKFWARGRKPWKLGIQLLKIAMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYM
DRMDDTYAVYTQTDVYDQIFFAINQYLQLPNISVGNHAYEKKGAEETALAVCQQFYKQGTICPGNDTFDI
DPEIVTDCLYIEPMMSLDNRTVGKHNLNFTLDFHRLVAVQLMFNLKAINLQTVRHHELPDCYDFTLTIVF
DNKAHSGRIKISLDNDIEIRECKDWHVSGSIQKNTHYMMIFDAFVILICLSSLILCTRSVVKGIRLQREF
VSFFLYYYKKEVSYNDQMEFVNGWYILIMVSDVLTIVGSTLKMEIQAKSLTSYDVCSILLGTSTMLVWLG
VIRYLGFFQKYNLLILTLRAALPNVMRFCCCAAMIYLGYCFCGWIVLGPYHVKFRSLNVVSECLFSLING
DDMFATFAKMQQKSYLVWLFSRIYLYSFISLFIYMVLSLFIALITDTYETIKHYQQDGFPETELQRFISQ
CKDLPNSGRYRLEEEGSVSLFCCCSGPSEHI (SEQ ID NO:12) Canis familiaris
TRPML3 (XM_547306) (SEQ ID NO:13)
ATGACCCCTTTTGGCAGCTTGGCTTCTGCAAAGGCTTCAAACTCAAGAGCTGCCTGGAAGATTGTGGTGG
ATTCATTCAGCTCGCAGCACACGCCCCAGGTGGGCACTGGTGGTCCCCAGGAATCAGACAAGGCCCTTAC
CTTCGGAGAGTTAACGTTCTCCCACTCATCTCCATTCTTCATCTGCGCCAGCCCTTCTCTCCCACCGTTG
CACAGCAGTGGGCTAGACGATGAGCCATACTGCTGGACAGGTTTTCACTGCATCAAGTACCTCGCGGGCC
CAGCGAGTGTCCCAAACTCCCTTGAAAGAGGGAGTAAGATATTGGTTTCCCAAGCCTCCTTTCCCATCCG
GACCTCCCCTTACCTGACACTGCTAAGCCGAGGGGAAAAGAAGCCTCTCTGCAGTTCCGTGGAGAAAAGG
CCTTTGGGGGTTTTGGAGATGGGAAGTCTGACTCTCCTCTCGGAAGAGCTCAAACGGCAGCTCCCCGGCA
CGCTGTTGCTAGGACAACCTCCGTTGCTAAGGGAAAGAGGCGGCTCCTCTGCTGAGATTGACAAGACGCC
GCTACCAATAGGGCGTCTACTCTCCGGCCGCCTCTTCAAGGCCGCACTTGTGATTGGCTGCTGTCGTGCT
GACGTCACGCAACTCGAACCGCCGAGAGATCCTGGGTTTTCGCCCGCTCGGCAGGAGGTGTGCGGTTTGG
GTTCCCGCGTGGAGGGTGCTGCTGGCTCGAATGTAAACAATCTTTTTTTTTTTCTCCCCCTAGAGATGGC
AAATCCTACGGTTGTTATAAGTAGCTGCAGCTCTCATGAAGAGGAAAATCGTTGCACTTTTAGCCAGCAC
ACATCGCCCTCTGAGGAGCTTCTGTTAGAAGACCAGATAAGGCGAAAACTCAAATTTTTTTTCATGAATC
CTTGTGAAAAGTTCTGGGCTCGAGGTAGAAAACCATGGAAGCTTGCCATACAAATTCTAAAAATTGCAAT
GGTGACTATCCAGCTGGTCTTTTTTGGGCTAAGTAACCAGATGGTTGTAGCTTTCAAGGAAGAAAACACT
ATAGCATTCAAACACCTCTTCTTAAAAGGATATATGGACCGAATGGATGACACATATGCAGTGTACACAC
AACGTGATGTATATGATCAGATCATCTTTGCAGTGAACCAGTACTTGCTTCTACGCAATACCTCGGTTGG
GAATCATGCTTATGAGAACAAGGGGACGGAACAGTCTGCTATGGCAATCTGTCAGCACTTCTACAAGCAG
GGAAACATCTGTCCTGGAAATGATACCTTCGACATTGATCCAGAGATTGAAACTGAGTGTTTCTCTGTAG
AGCCAGCTGAGCCTTTCCACGTCGGAACACTGGAAGAAAATAAACTCAACTTAACGCTGGACTTTCACAG
ACTCCTCACGGTGGACCTGCAGTTTAAGCTGAAGGCCATTAATCTGCAGACCATTCGGCATCACGAGCTC
CCTGACTGTTATGACTTTACTCTCACTATAACATTTGACAATAAGGCCCATAGTGGAAGAATTAAGATAA
GTTTAGATAATGATATTTCCATCAGAGAATGTAAAGACTGGCATGTATCGGGATCAATTCAGAAGAACAC
TCACTACATGATGATCTTTGATGCCTTTGTTATTCTGACATGCTTGGCTTCACTAACCCTGTGCCTTCGA
TCTGTAATTAGAGGACTTCAGCTTCAACAGGAATTTGTCAATTTTTTCCTCCTCCATTATAAGAAGGAAG
TTTCTGTTTCTGATCGAATGGAATTTGTCAATGGATGGTACATTATGATTATTATTAGTGACATGTTGAC
AATTATTGGATCAATTCTGAAAATGGAAATTCAAGCTAAGAGTCTAACAAGTTATGATGTTTGTAGCATA
CTTCTTGGGACTTCCACCATGCTTGTGTGGCTTGGAGTTATTCGATACCTCGGCTTCTTTCAGAAGTACA
ATCTCCTTATTCTGACCCTGCAGGCAGCACTGCCCAGTGTCATCAGGTTCTGTTGCTGTGCCGCTATGAT
TTATTTAGGCTATTGCTTCTGTGGATGGATTGTGCTGGGGCCGTACCATGATAAGTTCCGTTCTCTGAAC
ATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGATATGTTTGCCACATTTGCAAAAATGCAAC
AAAAAAGTTACTTGGTCTGGCTGTTTAGCAGAATTTATCTCTACTCATTCATCAGCCTCTTTATATATAT
GATTTTAAGTCTTTTCATCGCACTGATCACTGATACGTATGAAACAATTAAGCATTACCAACAAGATGGC
TTTCCAGAGACTGAACTTCGTACATTTATATCAGAGTGCAAAGATCTACCCAATTCTGGAAAATACAGAT
TAGAAGATGACACTCCAATATCTATTCTGCTGTTGTAAAAAG (SEQ ID NO:13) Canis
familiaris TRPML3 (XM_547306) (SEQ ID NO:14)
MTPFGSLASAKASNSRAAWKIVVDSFSSQHTPQVGTGGPQESDKALTFGELTFSHSSPFFICASPSLPPL
HSSGLDDEPYCWTGFHCIKYLAGPASVPNSLERGSKILVSQASFPIRTSPYLTLLSRGEKKPLCSSVEKR
PLGVLEMGSLTLLSEELKRQLPGTLLLGQPPLLRERGGSSAEIDKTPLPIGRLLSGRLFKAALVIGCCRA
DVTQLEPPRDPGFSPARQEVCGLGSRVEGAAGSNVNNLFFFLPLEMANPTVVISSCSSHEEENRCTFSQH
TSPSEELLLEDQIRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQLVFFGLSNQMVVAFKEENT
IAFKHLFLKGYMDRMDDTYAVYTQRDVYDQIIFAVNQYLLLRNTSVGNHAYENKGTEQSAMAICQHFYKQ
GNICPGNDTFDIDPEIETECFSVEPAEPFHVGTLEENKLNLTLDFHRLLTVDLQFKLKAINLQTIRHHEL
PDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQKNTHYMMIFDAFVILTCLASLTLCLR
SVIRGLQLQQEFVNFFLLHYKKEVSVSDRMEFVNGWYIMIIISDMLTIIGSILKEIQAKSLTSYDVCSIL
LGTSTMLVWLGVIRYLGFFQKYNLLILTLQAALPSVIRFCCCAAMIYLGYCFCGWIVLGPYHDKFRSLNM
VSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFIYMILSLFIALITDTYETIKHYQQDGF
PETELRTFISECKDLPNSGKYRLEDDTPISIFCCCKK (SEQ ID NO:14) Danio rerio
TRPML3 (XM_688418) (SEQ ID NO:15)
ATGTCTGACCGAGCGTCACACACTCATGAAAGCGCAACACTTCTGGACCCGGAGTGTGTGGAAAGCTTAA
GGAGAAAACTCAAGTATTTCTTCATGAGTCCGTGTCAGAAATACAGCACTAGAGGACGGATACCATGGAA
GATGATGCTTCAGATACTCAAGATTTGTTTAGTATTCATCTACCTGGTCTCTTTTGGATTGAGCAACGAG
ATGATGGTGACGTTCAAAGAGGAAAATCTCATCGCCTTCAAGCACTTCTTTCTGAAAAACTACAAGGACA
GCAATAAACATTACGCCTTGTACACAAAACATGAAGTTCACGACCACATCCTCTACACCATCAGACGGTA
TCTACAGCTACAAAACCTGACGATTGGCAATCAAGCGCTGGAGATGATCGATGGTCTGGCGACTCCTCTG
TCTCTCTGTCAGCAGTTGTATCGACATGCGCGCGTCGTGCCGTCTAATGAGACGTTTGAAATCGATCCAC
ATGTAGAGACAGAGTGTGTTTCTGTGTATCCCCTTTCTCCCATCACGACTGACAGTCTGGAAAACTCCCT
GAACTTGACTTTAGATTTTCAAAGGTTGTTAGCGGTAAACATTTATCTGAAGATCAAGGCTATCAACATT
CAGACGGTTCGCCATCAAGAGTTACCAGACTGCTACGACTTCAGCATTAATATCATGTTTGACAATCGTG
CACACAGCGGACAGATCAAGATCTCTCTCAGCAGCGGCGTGCAGATAAACGTCTGTAAGGACTGGAACAT
TTCTGGCTCAAGTAAGTTGAACAGCCACTTTGCGCTGATTGTGGTGTTTGACTGTTTGATCATCTGCTTC
TGTCTGCTGTCACTCATCCTCTGCACGCGCTCAGTCCACACAGGATTTCTCCTACAGACTGAATACAGAA
GATTCATGTCCAGTCAGCACAGTAAAAGCGTCTCATGGTCTGAGAGGCTGGAGTTCATCAACGGCTGGTA
CATCCTCATCATCATCAGCGATGCGCTGACTATTGCAGGCTCAATCCTCAAAATCTGCATACAGAGCAAA
GAACTGACGAGCTATGACGTGTGCAGTATTCTGCTGGGCACTGCAACAATGCTGGTGTGGATTGGAGTAA
TGCGCTACCTCAGTTTCTTCCAGAAATATTATATCCTCATCCTCACCCTGAAGGCTGCACTTCCCAATGT
GATTCGATTCTCCATCTGCGCTGTTATGATCTACCTGAGTTACTGCTTCTGCGGATGGATCGTTTTGGGG
CCACACCATGAAAATTTTCGCACATTCAGTAGGGTTGCTGGCTGTCTTTTCTCCATGATTAATGGGGATG
AAATCTACTCCACGTTCACCAAGCTCCGGGAATACAGCACTCTGGTGTGGCTGTTCAGCAGACTCTACGT
CTACAGCTTCATCCCGGTCTTCACATACATGGTTCTGAGTGTCTTCATCGCCCTCATCACAGACACGTAT
GAAACCATCAGGGTGAGTTATTTCAGCTTCAGTGAGAGTAGCTGCAAA (SEQ ID NO:15)
Danio rerio TRPML3 (XM_688418) (SEQ ID NO:16)
MSDRASHTHESATLLDPECVESLRRKLKYFFMSPCQKYSTRGRIPWKMMLQILKICLVFIYLVSFGLSNE
MMVTFKEENLIAFIKHFFLKNYKDSNKHYALYTKHEVHDHILYTIRRYLQLQNLTIGNQALEMIDGLATP
LSLCQQLYRHARVVPSNETFEIDPHVETECVSVYPLSPITTDSLENSLNLTLDFQRLLAVNIYLKIKAIN
IQTVRHQELPDCYDFSINIMFDNRAHSGQIKISLSSGVQINVCKDWNISGSSKLNSHFALIVVFDCLIIC
FCLLSLILCTRSVHTGFLLQTEYRRFMSSQHSKSVSWSERLEFINGWYILIIISDALTIAGSILKICIQS
KELTSYDVCSILLGTATMLVWIGVMRYLSFFQKYYILILTLKAALPNVIRFSICAVMIYLSYCFCGWIVL
GPHHENFRTFSRVAGCLFSMINGDEIYSTFTKLREYSTLVWLFSRLYVYSFIPVFTYMVLSVFIALITDT
YETIRVSYFSFSESSCK (SEQ ID NO:16) Homo sapiens TRPML3
Codon-optimized (SEQ ID NO:17)
ATGGCCGACCCTGAGGTGGTGGTGTCCTCCTGCTCTAGCCACGAGGAAGAGAACCGGTGCAACTTCAACC
AGCAGACCAGCCCCAGCGAGGAACTGCTGCTGGAAGATCAGATGCGGCGGAAGCTGAAGTTCTTCTTCAT
GAACCCCTGCGAGAAGTTCTGGGCCAGAGGCCGGAAGCCTTGGAAGCTGGCCATCCAGATCCTGAAGATC
GCCATGGTGACCATCCAGCTGGTGCTGTTCGGCCTGAGCAACCAGATGGTGGTGGCCTTCAAAGAGGAAA
ACACAATCGCCTTCAAGCACCTGTTTCTGAAGGGCTACATGGACCGGATGGACGACACCTACGCCGTGTA
CACCCAGAGCGACGTGTACGACCAGCTGATCTTCGCCGTGAACCAGTACCTGCAGCTGTACAACGTGAGC
GTGGGCAACCACGCCTACGAGAACAAGGGCACCAAGCAGAGCGCCATGGCCATCTGCCAGCACTTCTACA
AGCGGGGCAACATCTACCCCGGCAACGACACCTTCGACATCGACCCCGAGATCGAGACAGAGTGCTTCTT
CGTGGAGCCCGACGAGCCTTTCCACATCGGCACCCCTGCCGAGAACAAGCTGAACCTGACCCTGGACTTC
CACCGGCTGCTGACCGTGGAGCTGCAGTTCAAGCTGAAGGCCATCAACCTGCAGACCGTGCGGCACCAGG
AACTGCCCGACTGCTACGACTTCACCCTGACCATCACCTTCGATAACAAGGCCCACAGCGGCCGGATCAA
GATCAGCCTGGACAACGACATCAGCATCCGGGAGTGCAAGGACTGGCACGTGAGCGGCAGCATCCAGAAA
AACACCCACTACATGATGATCTTCGACGCCTTCGTGATCCTGACCTGCCTGGTGTCCCTGATCCTGTGCA
TCAGAAGCGTCATCAGGGGCCTGCAGCTCCAGCAGGAATTCGTCAACTTCTTCCTGCTGCACTACAAGAA
AGAAGTGTCCGTCAGCGACCAGATGGAATTTGTGAACGGCTGGTACATCATGATCATCATCAGCGACATC
CTGACAATCATCGGCAGCATTCTGAAGATGGAAATCCAGGCCAAGAGCCTGACCAGCTACGACGTGTGCA
GCATTCTGCTGGGAACCTCCACCATGCTGGTCTGGCTCGGCGTGATCAGATACCTGGGCTTCTTCGCCAA
GTACAACCTGCTGATTCTGACACTGCAGGCCGCCCTGCCCAACGTGATCCGGTTCTGCTGCTGCGCCGCC
ATGATCTACCTGGGCTACTGCTTCTGCGGCTGGATCGTGCTGGGCCCCTACCACGACAAGTTCCGGTCCC
TGAACATGGTGTCCGAGTGCCTGTTCAGCCTGATCAACGGCGACGACATGTTCGCCACCTTCGCCAAGAT
GCAGCAGAAAAGCTACCTGGTCTGGCTGTTCAGCCGGATCTACCTGTACAGCTTCATCAGCCTGTTCATC
TACATGATCCTGAGCCTGTTTATCGCCCTGATCACCGATACCTACGAGACAATCAAGCAGTACCAGCAGG
ACGGCTTCCCCGAGACAGAGCTGCGGACCTTCATCAGCGAGTGTAAGGACCTGCCCAACAGCGGCAAGTA
CCGGCTGGAAGATGACCCCCCCGTGTCCCTGTTCTGCTGTTGCAAGAAG (SEQ ID NO:17)
Homo sapiens TRPML3 CDS variant C1149T silent mutation (SEQ ID
NO:18)
ATGGCAGATCCTGAGGTAGTTGTGAGTAGCTGCAGCTCTCATGAAGAGGAAAATCGCTGCAATTTTAACC
AGCAAACATCTCCATCTGAGGAGCTTCTATTAGAAGACCAGATGAGGCGAAAACTCAAATTTTTTTTCAT
GAATCCCTGTGAGAAGTTCTGGGCTCGAGGTAGAAAACCATGGAAACTTGCCATACAAATTCTAAAAATT
GCAATGGTGACTATCCAGCTGGTCTTATTTGGGCTAAGTAACCAGATGGTGGTAGCTTTCAAGGAAGAGA
ATACTATAGCATTCAAACACCTTTTCCTAAAAGGATATATGGACCGAATGGATGACACATATGCAGTGTA
CACACAAAGTGACGTGTATGATCAGTTAATCTTCGCAGTAAACCAGTACTTGCAGCTATACAATGTCTCC
GTTGGGAATCATGCTTATGAGAACAAAGGTACCAAGCAATCTGCTATGGCAATCTGTCAGCACTTCTACA
AGCGAGGAAACATCTACCCTGGAAATGATACCTTTGACATCGATCCAGAAATTGAAACTGAGTGTTTCTT
TGTGGAGCCAGATGAACCTTTTCACATTGGGACACCAGCAGAAAATAAACTGAACTTAACACTGGACTTC
CACAGACTCCTAACAGTGGAGCTTCAGTTTAAACTGAAAGCCATTAATCTGCAGACAGTTCGTCATCAAG
AACTCCCTGACTGTTATGACTTTACTCTGACTATAACATTTGACAACAAGGCCCATAGTGGAAGAATTAA
AATAAGTTTAGATAATGACATTTCCATCAGAGAATGTAAAGACTGGCATGTATCTGGATCAATTCAGAAG
AACACTCATTACATGATGATCTTTGATGCCTTTGTCATTCTGACTTGCTTGGTTTCATTAATCCTCTGCA
TTAGATCTGTGATTAGAGGACTTCAGCTTCAGCAGGAGTTTGTCAATTTTTTCCTCCTCCATTATAAGAA
GGAAGTTTCTGTTTCTGATCAAATGGAATTTGTCAATGGATGGTACATTATGATTATTATTAGTGACATA
TTGACAATCATTGGATCAATTCTAAAAATGGAAATCCAAGCTAAGAGTCTAACTAGTTATGATGTCTGTA
GCATACTTCTTGGGACTTCTACCATGCTTGTGTGGCTTGGAGTCATCCGATACCTCGGTTTCTTTGCAAA
GTACAACCTCCTCATTTTGACCCTTCAGGCAGCGCTGCCCAATGTCATCAGGTTCTGCTGCTGTGCAGCT
ATGATTTACTTAGGTTACTGCTTCTGTGGATGGATCGTGCTGGGGCCTTACCATGACAAGTTTCGTTCTC
TGAACATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGATATGTTTGCCACGTTTGCAAAAAT
GCAGCAAAAAAGTTACTTAGTCTGGCTGTTTAGTAGAATTTACCTCTACTCATTCATCAGCCTCTTTATA
TATATGATTTTAAGTCTTTTCATTGCACTGATCACTGATACATACGAAACAATTAAGCAATACCAACAAG
ATGGCTTCCCAGAGACTGAACTTCGTACATTTATATCAGAATGCAAAGATCTACCCAACTCTGGAAAATA
CAGATTAGAAGATGACCCTCCAGTATCTTTATTCTGCTGTTGTAAAAAG (SEQ ID NO:18)
Rattus norevigus (NM_00102059.1) (GI:58865683) (SEQ ID NO:19)
gttcgacaga agcttgtgat ttatggtcca aggaatccag tgtcagatca atagacaaaa
61 tgccccaggg aagttgtgtg tgcattctac tggacagatc agagactggt
cagaacaggt 121 gcttggctgg cggtgcgtcc aaacctcaga gatggcaaat
cctgaggtgg tggtaagcag 181 ctgcagttct caccaggatg aaagtccctg
cactttctac ccgagctcat cccagtccga 241 gcagcttctc ttagaagatc
agatgaggcg gaaactcaaa ttctttttta tgaatccttg 301 cgagaagttc
tgggctcggg gtaggaagcc atggaaactt gccatacaga ttctgaaaat 361
cgctatggtg actatccagc tggttctgtt tggactaagt aaccagatgg tagtagcttt
421 caaggaagag aacacgatag ccttcaaaca cctcttcctg aaaggctaca
tggaccgaat 481 ggacgacacc tacgcggtgt acactcagaa tgatgtgtac
gaccagatca tctttgcagt 541 gacccggtac ttgcaacttc gaaacatctc
cgtcggcaac catgcttatg agaacaaggg 601 gactaagcag tcagcaatgg
cagtctgtca gcacttctac aggcaaggca ccatctgccc 661 cgggaacgat
accttcgaca tcgatccaga agtcgaaaca gactgtttcc ttatagagcc 721
agaggaagct ttccacatgg gaacacctgg agaaaacaaa ctcaacctga ccctggactt
781 ccacagactt ctgacagtgg agctccaatt taagctcaaa gccatcaacc
tgcagacagt 841 tcgccaccag gagcttcctg actgttacga ctttaccctg
actataacat tcgacaacaa 901 ggcacacagt ggaagaatca aaataagttt
agacaacgac atttctatca gagaatgcaa 961 agattggcac gtgtctggat
caattcagaa gaacacccac tacatgatga tcttcgatgc 1021 ctttgttatc
ctgacctgct tgtcctcgct ggtgctctgc gccaggtctg tgattcgggg 1081
tcttcagctt cagcaggagt ttgtcaactt tttccttctt cactacaaga aggaagtttc
1141 ggcctctgat cagatggagt tcatcaacgg gtggtacatt atgatcatcg
ttagtgacat 1201 actgacgatc gttggatcga ttctgaaaat ggaaatccaa
gccaagagtc ttacaagcta 1261 cgatgtctgt agcatacttc ttgggacttc
caccatgctc gtgtggcttg gcgttatccg 1321 atacctgggt ttctttgcga
agtacaatct ccttatcctg accctccagg cagcgctgcc 1381 caatgtcatc
aggttctgtt gctgtgcggc tatgatctat cttgggtatt gcttttgcgg 1441
atggattgtg ctgggccctt accatgagaa gttccgctct ctgaacaagg tctctgagtg
1501 cctattctca ctgataaatg gagacgacat gttttccacg ttcgcgaaaa
tgcagcagaa 1561 aagttacctg gtgtggctgt tcagccgcgt ctacctgtac
tcgttcatca gcctcttcat 1621 ctacatgatc ttgagccttt tcatcgcgct
catcacagac acgtacgaaa ccattaagca 1681 ctaccagcaa gatggcttcc
cagagacgga acttcgaaag tttatcgctg aatgcaaaga 1741 cctccccaac
tctggaaaat acagattgga agatgaccct ccaggttctt tattctgctg 1801
ctgcaaaaag taactgtcgg gtttctctgt gcctgggagg aaaatacagt gtgtggatga
1861 gtcagagaca atatggatta ttggtaatca cgcaacagtg tgttcagata
ctagtgttct 1921 gagttaactc acagctatga ctttgcgggg cctgttaaat
atatttttaa atattaaaaa 1981 aaaaaaaaaa aaaaaaaaaa Rattus norevigus
(NP_00102059.1) (GI:58865684) (SEQ ID NO:20)
MANPEVVVSSCSSHQDESPCTFYPSSSQSEQLLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKI
AMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQNDVYDQIIFAVTRYLQLRNIS
VGNHAYENKGTKQSAMAVCQHFYRQGTICPGNDTFDIDPEVETDCFLIEPEEAFHMGTPGENKLNLTLDF
HRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQK
NTHYMMIFDAFVILTCLSSLVLCARSVIRGLQLQQEFVNFFLLHYKKEVSASDQMEFINGWYIMIIVSDI
LTTVGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCAA
MIYLGYCFCGWIVLGPYHEKFRSLNKVSECLFSLINGDDMFSTFAKMQQKSYLVWLFSRVYLYSFISLFI
YMILSLFIALITDTYETIKHYQQDGFPETELRKFIAECKDLPNSGKYRLEDDPPGSLFCCCKK Sus
scrofa (AB271930) (GI:146741299) (Partial) (SEQ ID NO:21) 1
tgaaatggc agatcctgag cctgtcataa gtagctgcag ctctcgtgaa gaggaaaatc 61
gctgcacttt taaccagcac acatgtccct ctgaggagcg tctattagaa gaccagatga
121 ggcgaaaact caaatttttt ttcatgactc cttgtgagaa gttctggact
cgaggtcgaa 181 aaccatggaa acttgccatg caagttctaa aaattgcgat
ggtgactatc cagctgatct 241 ttttcgggct aagtaaccag atggtggtag
ctttcaagga agagaacacg atagcattta 301 aacacctctt tctaaagggc
tatgtggacc agatggatga cacatatgcc gtgtacaccc 361 aaagcgacgt
atacgatcgg atcgtcttcg cagtgaacca gtacttgcag ctacgcagca 421
tctcggttgg gaaccacgct tacgagaaca agggcgcgga gcagtcggcc atggcgatct
481 gttggcactt ctacaagcaa ggaaacatct gtcctggaaa tgacaccttt
gacgttgatc 541 cagaagtaaa aactgaatgt ttctttgttg agccggatga
agctgttgac actggaacac 601 tggaggagaa taagctcaac ttaacccttg
actttcacag actcctaacg gtggagctgc 661 agtttaaact caaggccatt
aatctgcaga cgattcgcca tcacgaactc cctgactgtt 721 atgacttcac
cctgaccata acatttgaca a Sus scrofa (BAF62305.1) (GI:14671300)
(Partial) (SEQ ID NO:22)
MADPEPVISSCSSREEENRCTFNQHTCPSEERLLEDQMRRKLKFFFMTPCEKFWTRGRKPWKLAMQVLKI
AMVTIQLIFFGLSNQMVVAFKEENTIAFKHLFLKGYVDQMDDTYAVYTQSDVYDRIVFAVNQYLQLRSIS
VGNHAYENKGAEQSAMAICWHFYKQGNICPGNDTFDVDPEVKTECFFVEPDEAVDTGTLEENKLNLTLDF
HRLLTVELQFKLKAINLQTIRHHELPDCYDFTLTITFD Pan troglytes TRPML3
(XM_001140555.1) (GI:114557485) (SEQ ID NO:23) 1 cttactccaa
tcaagcctct gcccgccagg aataggtaac ctgtgtgtgt ccgtttgctc 61
cttctaagag catgcctgat agatacttcg gtagcctctc cggatggccc cttcgtcggg
121 tagcctctcc tgatggggtc cttcgcccac cctgcctccc gcgccggcgc
tccgggtgaa 181 tgtcaagggt ggctggctgc gaatacctcc ttcagctgct
ggggttcccg acagtttgca 241 gtttttaaaa gtgcaccctc ggaagggctt
ttcagactgg gtaaagctga cttttccaag 301 agatggcaga tcctgaggta
gttgtgagta gctgcagctc tcatgaagag gaaaatcgct 361 gcaattttaa
ccagcaaaca tctccatctg aggagcttct attagaagac cagatgaggc 421
gaaaactcaa attttttttc atgaatccct gtgagaagtt ctgggctcga ggtagaaaac
481 catggaaact tgccatacaa attctaaaaa ttgcaatggt gactatccag
ctggtcttat 541 ttgggctaag taaccagatg gtggtagctt tcaaggaaga
gaatactata gcattcaaac 601 accttttcct aaaaggatat atggaccgaa
tggatgacac atatgcagtg tacacacaaa 661 gtgacgtgta tgatcagtta
atcttcgcag taaaccagta cttgcagcta tacaatgtct 721 ccgttgggaa
tcatgcttat gagaacaaag gtaccaagca atctgctatg gcaatctgtc 781
agcacttcta caagcgagga aacatctacc ctggaaatga tacctttgac atcgatccag
841 aaattgaaac tgagtgtttc tttgtggagc cagatgaacc ttttcacatt
gggacaccag 901 cagaaaataa actgaactta acactggact tccacagact
cctaacagtg gagcttcagt 961 ttaaactgaa agccattaat ctgcagacag
ttcgtcatca agaactccct gactgttatg 1021 actttactct gactataaca
tttgacaaca aggcccatag tggaagaatt aaaataagtt 1081 tagataatga
catttccatc agagaatgta aagactggca tgtatctgga tcaattcaga 1141
agaacactca ttacatgatg atctttgatg cctttgtcat tctgacttgc ttggtttcat
1201 taatcctctg cattagatct gtgattagag gacttcagct tcagcaggta
gggaacgttg 1261 ctttctagga atgctactga cattttgatt gacagagaca
ttcactgtgc ctcccctctt 1321 ttccctaaag gagtttgtca attttttcct
cctccattat aagaagggag tttctgtttc 1381 tgatcaaatg gaatttgtca
atggatggta cattatgatt attattagtg acatattgac 1441 aatcattgga
tcaattctaa aaatggaaat ccaagctaag gtaatttttt tcctaatcat 1501
gctattgtta gtgtcagatt tgcactaatg gtaatgtatt tttccagaat gtaagaattt
1561 tcagaatgaa ttgtttcttc caaactgtat atcaagtaga cttgaaattg
gtaatggtaa 1621 ttttcttaaa tctagtcagg aggtctctta ggcagagttt
ttcaaagtgt gatccacaaa 1681 ccattgcatc agaatcattg ggtgcctggt
aaagtgtacc atgttagacc tactgaattc 1741 agactcttcg gcggggcctg
tgaattctta cacacaccaa aattcataca caaccaaggt 1801 aactaaggta
agagtttttt ttttttttaa atcttacaag aaatgctcaa atctttaaca 1861
aaaatgagtg ggtctatagg ggaaagtgag gtcaaggcac tatggtgtgc atgcttgcat
1921 ttgtttcctc cgtccattca aagtgagaat gctcccattt tcttacttta
ccattgatgt 1981 gctacaagct tatttatttt aagactaacc tagcctaaaa
atcaactgtc cccacaaaat 2041 aaaaatcaca ttaaaaaaac taatagtgtt
cagactaatc ttgctcaaac ttatgtttcc 2101 ctagtcttga tgcgactgat
tgagtcacct ggggagttgg ttataaacct gggcagagac 2161 cccaaatgca
atggctcaga gaagatagga gcttatttct gtcttatgca atagtcagaa 2221
tgggttttac agactggtga gtagctcaac atctcacagt cattcaggca cccatgttcc
2281 tcccattttg tttctctgcc atcccttaag gacttgccct gactgcatga
ttattgctgt 2341 gttgcctcaa acaggttgca gcttatggga agcaaaaaca
cggtatggtg gaagctctcc
2401 catagactga tggcttggct caagagtggc cgactttatt tctgtacata
tcccactgga 2461 tagaatttag tcaatcctaa ctgcagaggg agccagggaa
cacagcccag gcatgtgcct 2521 aggaagggga gaatgggttt aggttgacac
ttagcagctg ccactatatg tggctatagt 2581 atgtatcatt ggaatagatg
tttaacttta gggacaaata aacaaaaaac caaaacaaaa 2641 aaaggagtaa
ggggagagat ttgcagcaaa tctttatttt taccaacctc aactatcatt 2701
aatttcagtg aaccctaaat ggtgtccaac aaaatatctt tctagaccat tcaccgtctc
2761 tgcctcatag atgatcatat catgttttct tctcttctga aacctctaat
acccttgtcc 2821 tatcctcatt ctaagctgat gaccttactt cctatttcac
aaaaataata gaaaaaaaaa Pan troglytes TRPML3 (XP_001140555.1)
(GI:469371) (SEQ ID NO:24)
MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKI
AMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVS
VGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDF
HRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQK
NTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQVGNVAF Pan troglytes TRPML3
(XM_001140401.1) (GI:114557483) (SEQ ID NO:25) 1 cctctagaga
tggcagatcc tgaggtagtt gtgagtagct gcagctctca tgaagaggaa 61
aatcgctgca attttaacca gcaaacatct ccatctgagg agcttctatt agaagaccag
121 atgaggcgaa aactcaaatt ttttttcatg aatccctgtg agaagttctg
ggctcgaggt 181 agaaaaccat ggaaacttgc catacaaatt ctaaaaattg
caatggtgac tatccagctg 241 gtcttatttg ggctaagtaa ccagatggtg
gtagctttca aggaagagaa tactatagca 301 ttcaaacacc ttttcctaaa
aggatatatg gaccgaatgg atgacacata tgcagtgtac 361 acacaaagtg
acgtgtatga tcagttaatc ttcgcagtaa accagtactt gcagctatac 421
aatgtctccg ttgggaatca tgcttatgag aacaaaggta ccaagcaatc tgctatggca
481 atctgtcagc acttctacaa gcgaggaaac atctaccctg gaaatgatac
ctttgacatc 541 gatccagaaa ttgaaactga gtgtttcttt gtggagccag
atgaaccttt tcacattggg 601 acaccagcag aaaataaact gaacttaaca
ctggacttcc acagactcct aacagtggag 661 cttcagttta aactgaaagc
cattaatctg cagacagttc gtcatcaaga actccctgac 721 tgttatgact
ttactctgac tataacattt gacaacaagg cccatagtgg aagaattaaa 781
ataagtttag ataatgacat ttccatcaga gaatgtaaag actggcattc tccctccgtc
841 gcccagcctg gaaacactca ttacatgatg atctttgatg cctttgtcat
tctgacttgc 901 ttggtttcat taatcctctg cattagatct gtgattagag
gacttcagct tcagcaggag 961 tttgtcaatt ttttcctcct ccattataag
aagggagttt ctgtttctga tcaaatggaa 1021 tttgtcaatg gatggtacat
tatgattatt attagtgaca tattgacaat cattggatca 1081 attctaaaaa
tggaaatcca agctaagagt ctaactagtt atgatgtctg tagcatactt 1141
cttgggactt ctaccatgct tgtgtggctt ggagtcatcc gatacctcgg tttctttgca
1201 aagtacaatc tcctcatttt gacccttcag gcagcactgc ccaatgtcat
caggttctgc 1261 tgctgtgcag ctatgattta cttaggttac tgcttctgtg
gatggatcgt gctggggcct 1321 taccatgaca agtttcgttc tctgaacatg
gtctctgagt gccttttctc tctgataaat 1381 ggagatgata tgtttgccac
gtttgcaaaa atgcagcaaa aaagttactt agtctggctg 1441 tttagtagaa
tttacctcta ctcattcatc agcctcttta tatatatgat tttaagtctt 1501
ttcattgcac tgatcactga tacatacgaa acaattaagc aataccaaca agatggcttc
1561 ccagagactg aacttcgtac atttatatca gaatgcaaag atctacccaa
ctctggaaaa 1621 tacagattag aagatgaccc tccagtatct ttattctgct
gttgtaaaaa gtagctatca 1681 ggtttatctg tactttagag gaaaatataa
tgtgtagctg agttagaaca ctgtggatat 1741 tctgagatca gatgtagtat
gtttgaagac tgttattttg agctaattga gacctataat 1801 tcaccaataa
ctgtttatat ttttaaaagc aatatttaat gtctttgcag ctttatgctg 1861
ggcttgtt Pan troglytes TRPML3 XP_001140401.1 (GI:114557484) (SEQ ID
NO:26)
MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKI
AMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVS
VGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDF
HRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHSPSVAQP
GNTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKGVSVSDQMEFVNGWYIMIIISD
ILTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCA
AMIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLF
IYMILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVSLFCCCKK
Pan troglytes TRPML3 (XM_524756.2) (GI:114557481) (SEQ ID NO:27) 1
tcctctagag atggcagatc ctgaggtagt tgtgagtagc tgcagctctc atgaagagga
61 aaatcgctgc aattttaacc agcaaacatc tccatctgag gagcttctat
tagaagacca 121 gatgaggcga aaactcaaat tttttttcat gaatccctgt
gagaagttct gggctcgagg 181 tagaaaacca tggaaacttg ccatacaaat
tctaaaaatt gcaatggtga ctatccagta 241 cttgcagcta tacaatgtct
ccgttgggaa tcatgcttat gagaacaaag gtaccaagca 301 atctgctatg
gcaatctgtc agcacttcta caagcgagga aacatctacc ctggaaatga 361
tacctttgac atcgatccag aaattgaaac tgagtgtttc tttgtggagc cagatgaacc
421 ttttcacatt gggacaccag cagaaaataa actgaactta acactggact
tccacagact 481 cctaacagtg gagcttcagt ttaaactgaa agccattaat
ctgcagacag ttcgtcatca 541 agaactccct gactgttatg actttactct
gactataaca tttgacaaca aggcccatag 601 tggaagaatt aaaataagtt
tagataatga catttccatc agagaatgta aagactggca 661 tgtatctgga
tcaattcaga agaacactca ttacatgatg atctttgatg cctttgtcat 721
tctgacttgc ttggtttcat taatcctctg cattagatct gtgattagag gacttcagct
781 tcagcaggag tttgtcaatt ttttcctcct ccattataag aagggagttt
ctgtttctga 841 tcaaatggaa tttgtcaatg gatggtacat tatgattatt
attagtgaca tattgacaat 901 cattggatca attctaaaaa tggaaatcca
agctaagagt ctaactagtt atgatgtctg 961 tagcatactt cttgggactt
ctaccatgct tgtgtggctt ggagtcatcc gatacctcgg 1021 tttctttgca
aagtacaatc tcctcatttt gacccttcag gcagcactgc ccaatgtcat 1081
caggttctgc tgctgtgcag ctatgattta cttaggttac tgcttctgtg gatggatcgt
1141 gctggggcct taccatgaca agtttcgttc tctgaacatg gtctctgagt
gccttttctc 1201 tctgataaat ggagatgata tgtttgccac gtttgcaaaa
atgcagcaaa aaagttactt 1261 agtctggctg tttagtagaa tttacctcta
ctcattcatc agcctcttta tatatatgat 1321 tttaagtctt ttcattgcac
tgatcactga tacatacgaa acaattaagc aataccaaca 1381 agatggcttc
ccagagactg aacttcgtac atttatatca gaatgcaaag atctacccaa 1441
ctctggaaaa tacagattag aagatgaccc tccagtatct ttattctgct gttgtaaaaa
1501 gtagctatca ggtttatctg tactttagag gaaaatataa tgtgtagctg
agttagaaca 1561 ctgtggatat tctgagatca gatgtagtat gtttgaagac
tgttattttg agctaattga 1621 gacctataat tcaccaataa ctgtttatat
ttttaaaagc aatatttaat gtctttgcag 1681 ctttatgctg ggcttgtttt
taaaacaact ttaatgagga aagctattgg attattatta 1741 tttcttgttt
attttgccat ggctttagaa tgtattctgt atgcctctct tttgctctga 1801
tactcttgct tcctgctatt ctgattgtgc agactgtgta attagtggaa aacaatcctt
1861 ggtctgactg tgactttgga caactcagta accctggctt ggaccactct
caggagtcca 1921 tccttgagag agtgggtgta gttaccattt atacagtaat
cattgcattt taaaatctgc 1981 tcttgaaagg aagaataaga gtgcaccaga
ataagagcgc accagaataa gagcgcacca 2041 gctaacaatg tgatacggcc
atatgtcact taaggataga gatatgttct gagaaatgtg 2101 tcattaggcg
attttgtcat taaacatcat agcatgtact tccacaaacc tagatggtat 2161
agcctactac acacctaggc tatttggtat agcctgttga tcctggggta caaatctgta
2221 caacatgtta ctgtattgaa tacagtaggc aattgtaaca caatggtaag
tatctaaaca 2281 tagaaaaggg acagtaaaaa tatggtttta taatcttctg
ggaccaccat tgtatatgcg 2341 gtacatcgtt gaccaaaaca tcgttatcca
gcatatgact gtatttggtt atgaaagcca 2401 actgttactt gattctgctt
ttagttctta agaggatcag gtttttaaat actcatttac 2461 aagttttcta
tcctccttca gtgttaaagt agaaagtaaa aagagtatct tatacatgca 2521
tgaaattaaa gcatatacca aatgca Pan troglytes TRPML3 (XP_524756.2)
(GI:114557482) (SEQ ID NO:28)
MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKI
AMVTIQYLQLYNVSVGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHI
GTPAENKLNLTLDFHRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISI
RECKDWHVSGSIQKNTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKGVSVSDQME
FVNGWYIMIIISDILTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQ
AALPNVIRFCCCAAMIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWL
FSRIYLYSFISLFIYMILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVS
LFCCCKK Pan troglytes TRPML3 (XM_00114082.1) (GI:114557479) (SEQ ID
NO:29) 1 tcctctagag atggcagatc ctgaggtagt tgtgagtagc tgcagctctc
atgaagagga 61 aaatcgctgc aattttaacc agcaaacatc tccatctgag
gagcttctat tagaagacca 121 gatgaggcga aaactcaaat tttttttcat
gaatccctgt gagaagttct gggctcgagg 181 tagaaaacca tggaaacttg
ccatacaaat tctaaaaatt gcaatggtga ctatccagct 241 ggtcttattt
gggctaagta accagatggt ggtagctttc aaggaagaga atactatagc 301
attcaaacac cttttcctaa aaggatatat ggaccgaatg gatgacacat atgcagtgta
361 cacacaaagt gacgtgtatg atcagttaat cttcgcagta aaccagtact
tgcagctata 421 caatgtctcc gttgggaatc atgcttatga gaacaaaggt
accaagcaat ctgctatggc 481 aatctgtcag cacttctaca agcgaggaaa
catctaccct ggaaatgata cctttgacat 541 cgatccagaa attgaaactg
agtgtttctt tgtggagcca gatgaacctt ttcacattgg 601 gacaccagca
gaaaataaac tgaacttaac actggacttc cacagactcc taacagtgga 661
gcttcagttt aaactgaaag ccattaatct gcagacagtt cgtcatcaag aactccctga
721 ctgttatgac tttactctga ctataacatt tgacaacaag gcccatagtg
gaagaattaa 781 aataagttta gataatgaca tttccatcag agaatgtaaa
gactggcatg tatctggatc 841 aattcagaag aacactcatt acatgatgat
ctttgatgcc tttgtcattc tgacttgctt 901 ggtttcatta atcctctgca
ttagatctgt gattagagga cttcagcttc agcaggagtt 961 tgtcaatttt
ttcctcctcc attataagaa gggagtttct gtttctgatc aaatggaatt 1021
tgtcaatgga tggtacatta tgattattat tagtgacata ttgacaatca ttggatcaat
1081 tctaaaaatg gaaatccaag ctaagagtct aactagttat gatgtctgta
gcatacttct 1141 tgggacttct accatgcttg tgtggcttgg agtcatccga
tacctcggtt tctttgcaaa 1201 gtacaatctc ctcattttga cccttcaggc
agcactgccc aatgtcatca ggttctgctg 1261 ctgtgcagct atgatttact
taggttactg cttctgtgga tggatcgtgc tggggcctta 1321 ccatgacaag
tttcgttctc tgaacatggt ctctgagtgc cttttctctc tgataaatgg 1381
agatgatatg tttgccacgt ttgcaaaaat gcagcaaaaa agttacttag tctggctgtt
1441 tagtagaatt tacctctact cattcatcag cctctttata tatatgattt
taagtctttt 1501 cattgcactg atcactgata catacgaaac aattaagcaa
taccaacaag atggcttccc 1561 agagactgaa cttcgtacat ttatatcaga
atgcaaagat ctacccaact ctggaaaata 1621 cagattagaa gatgaccctc
cagtatcttt attctgctgt tgtaaaaagt agctatcagg 1681 tttatctgta
ctttagagga aaatataatg tgtagctgag ttagaacact gtggatattc 1741
tgagatcaga tgtagtatgt ttgaagactg ttattttgag ctaattgaga cctataattc
1801 accaataact gtttatattt ttaaaagcaa tatttaatgt ctttgcagct
ttatgctggg 1861 cttgttttta aaacaacttt aatgaggaaa gctattggat
tattattatt tcttgtttat 1921 tttgccatgg ctttagaatg tattctgtat
gcctctcttt tgctctgata ctcttgcttc 1981 ctgctattct gattgtgcag
actgtgtaat tagtggaaaa caatccttgg tctgactgtg 2041 actttggaca
actcagtaac cctggcttgg accactctca ggagtccatc cttgagagag 2101
tgggtgtagt taccatttat acagtaatca ttgcatttta aaatctgctc ttgaaaggaa
2161 gaataagagt gcaccagaat aagagcgcac cagaataaga gcgcaccagc
taacaatgtg 2221 atacggccat atgtcactta aggatagaga tatgttctga
gaaatgtgtc attaggcgat 2281 tttgtcatta aacatcatag catgtacttc
cacaaaccta gatggtatag cctactacac 2341 acctaggcta tttggtatag
cctgttgatc ctggggtaca aatctgtaca acatgttact 2401 gtattgaata
cagtaggcaa ttgtaacaca atggtaagta tctaaacata gaaaagggac 2461
agtaaaaata tggttttata atcttctggg accaccattg tatatgcggt acatcgttga
2521 ccaaaacatc gttatccagc atatgactgt atttggttat gaaagccaac
tgttacttga 2581 ttctgctttt agttcttaag aggatcaggt ttttaaatac
tcatttacaa gttttctatc 2641 ctccttcagt gttaaagtag aaagtaaaaa
gagtatctta tacatgcatg aaattaaagc 2701 atataccaaa tgca Pan troglytes
TRPML3 (XP_00114082.1) (GI:114557480) (SEQ ID NO:30)
MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKI
AMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVS
VGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDF
HRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQK
NTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKGVSVSDQMEFVNGWYIMIIISDI
LTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCAA
MIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFI
YMILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVSLFCCCKK
Xenopus tropicalis (Silurana tropicalis) (NM_001016804.2)
(GI:77682897) (SEQ ID NO:31) 1 ttgcaactag gtctgacagt aggacaatgt
ggcaggtcac gtgacagcag tgctgatggt 61 agagatgcgc cagcattcag
gtctgagagc agaaagaaaa gctggccaaa acaaaggaca 121 ttctctttgc
tgcttcgcta gctgagacgc tgctatagta tagcagacat ggaaaaccca 181
gagctaataa agacatgcaa ctctttggat gaacatgatg gtccctactg ctgcaagcag
241 tgccctatga ctgatgagct acttatggaa gaccagctac gaaggaaact
taaattcttt 301 ttcatgaacc catgtgagaa gttccgtgcc cgtggacgaa
agccttggaa gctttgtatt 361 caaattttaa aaattgcaat ggtgacaatc
caattagttt tatttggact cagtaatgaa 421 atggtagtca cctttaaaga
ggagaacact gtagctttta agcatctgtt tttgaaagga 481 tataaggatg
gacatgatga cacatatgct atctacagtc aagaagatgt tcatgctcat 541
ataaacttta caattaaaag gtacctagag ctacaaaaca tatctgttgg aaatcatgca
601 tatgaaagta atggtaaagg tcaaactgga atgtcattat gtcaacatta
ctataaacaa 661 gggagtatct ttcctggaaa tgaaacattt gaaattgacc
cacaaataga tactgaatgt 721 ttccatattg atccatcaac tctgtgttct
aatgacacac ctgcagaata ctactggtct 781 aatatgacac tagacttcta
tagacttgtt tcagttgaaa ttatgtttaa gcttaaagca 841 attaatcttc
aaaccattcg tcatcatgaa cttccagact gctatgactt catggttata 901
ataacatttg ataataaggc acacagtgga aggataaaaa tcagcttaga taatgatgtt
961 ggaatccagg aatgcaaaga ctggcatgtg tctggatcta ttcaaaaaaa
tactcattac 1021 atgatgattt ttgatgctgc tgttattttg gtctgcttat
cttccataac actctgcatt 1081 cgctccgtgg ttaaaggaat tcacctacaa
aaagaatatg taaacttttt ccagcatcgt 1141 tttgcaagga ctgtgtcctc
agctgatcgc atggaatttg tcaatggctg gtacattatg 1201 ataatcatca
gtgatgtttt gtcaattatt ggctcaatct tgaagatgga gatccaagca 1261
aagagcctca ccagttatga tgtctgcagt attctcttgg gaacatccac cttattagtg
1321 tggcttggag ttattcgcta cttgggattt tttaagaaat acaatcttct
gatcctgaca 1381 cttagggcag ccttacctaa tgtgatacga ttctgctgtt
gtgctgctat gatctacctg 1441 ggctactgct tctgtggctg gattgttctg
gggccttacc atgtaaagtt caggtccctg 1501 aacatggttt cagagtgcct
gttctccctt attaatggag acgatatgtt tacaacgttt 1561 tcaatcatgc
aggagaagag ctacttggtt tggctgttta gtcgcattta tttgtattcc 1621
tttataagtc tcttcatata catggttctg agtctcttca ttgcacttat tactgacaca
1681 tacgatacaa tcaagaatta ccagatcgat ggctttccag aatcagaact
tcacacattt 1741 gtatccgagt gcaaagattt gccaacctct ggtcgatata
gggaacaaga cgagacctcc 1801 tgtttgtcta tgctgtgttg taatcggtaa
aaaagaatcc cagaagaagc actttatcca 1861 tggcctttaa aaatctgcaa
aaaaaaaaaa aaaaaa Xenopus tropicalis (Silurana tropicalis)
(NP_001016804.2) (GI:62857333) (SEQ ID NO:32)
MENPELIKTCNSLDEHDGPYCCKQCPMTDELLMEDQLRRKLKFFFMNPCEKFRARGRKPWKLCIQILKIA
MVTIQLVLFGLSNEMVVTFKEENTVAFKHLFLKGYKDGHDDTYAIYSQEDVHAHINFTIKRYLELQNISV
GNHAYESNGKGQTGMSLCQHYYKQGSIFPGNETFEIDPQIDTECFHIDPSTLCSNDTPAEYYWSNMTLDF
YRLVSVEIMFKLKAINLQTIRHHELPDCYDFMVIITFDNKAHSGRIKISLDNDVGIQECKDWHVSGSIQK
NTHYMMIFDAAVILVCLSSITLCIRSVVKGIHLQKEYVNFFQHRFARTVSSADRMEFVNGWYIMIIISDV
LSIIGSILKMEIQAKSLTSYDVCSILLGTSTLLVWLGVIRYLGFFKKYNLLILTLRAALPNVIRFCCCAA
MIYLGYCFCGWIVLGPYHVKFRSLNMYSECLFSLINGDDMFTTFSIMQEKSYLVWLFSRIYLYSFISLFI
YMVLSLFIALITDTYDTIKNYQIDGFPESELHTFVSECKDLPTSGRYREQDETSCLSMLCCNR
Xenopus tropicalis (Silurana tropicalis) (CR855599.2) (GI:77623768)
(SEQ ID NO:33) 1 ttgcaactag gtctgacagt aggacaatgt ggcaggtcac
gtgacagcag tgctgatggt 61 agagatgcgc cagcattcag gtctgagagc
agaaagaaaa gctggccaaa acaaaggaca 121 ttctctttgc tgcttcgcta
gctgagacgc tgctatagta tagcagacat ggaaaaccca 181 gagctaataa
agacatgcaa ctctttggat gaacatgatg gtccctactg ctgcaagcag 241
tgccctatga ctgatgagct acttatggaa gaccagctac gaaggaaact taaattcttt
301 ttcatgaacc catgtgagaa gttccgtgcc cgtggacgaa agccttggaa
gctttgtatt 361 caaattttaa aaattgcaat ggtgacaatc caattagttt
tatttggact cagtaatgaa 421 atggtagtca cctttaaaga ggagaacact
gtagctttta agcatctgtt tttgaaagga 481 tataaggatg gacatgatga
cacatatgct atctacagtc aagaagatgt tcatgctcat 541 ataaacttta
caattaaaag gtacctagag ctacaaaaca tatctgttgg aaatcatgca 601
tatgaaagta atggtaaagg tcaaactgga atgtcattat gtcaacatta ctataaacaa
661 gggagtatct ttcctggaaa tgaaacattt gaaattgacc cacaaataga
tactgaatgt 721 ttccatattg atccatcaac tctgtgttct aatgacacac
ctgcagaata ctactggtct 781 aatatgacac tagacttcta tagacttgtt
tcagttgaaa ttatgtttaa gcttaaagca 841 attaatcttc aaaccattcg
tcatcatgaa cttccagact gctatgactt catggttata 901 ataacatttg
ataataaggc acacagtgga aggataaaaa tcagcttaga taatgatgtt 961
ggaatccagg aatgcaaaga ctggcatgtg tctggatcta ttcaaaaaaa tactcattac
1021 atgatgattt ttgatgctgc tgttattttg gtctgcttat cttccataac
actctgcatt 1081 cgctccgtgg ttaaaggaat tcacctacaa aaagaatatg
taaacttttt ccagcatcgt 1141 tttgcaagga ctgtgtcctc agctgatcgc
atggaatttg tcaatggctg gtacattatg 1201 ataatcatca gtgatgtttt
gtcaattatt ggctcaatct tgaagatgga gatccaagca
1261 aagagcctca ccagttatga tgtctgcagt attctcttgg gaacatccac
cttattagtg 1321 tggcttggag ttattcgcta cttgggattt tttaagaaat
acaatcttct gatcctgaca 1381 cttagggcag ccttacctaa tgtgatacga
ttctgctgtt gtgctgctat gatctacctg 1441 ggctactgct tctgtggctg
gattgttctg gggccttacc atgtaaagtt caggtccctg 1501 aacatggttt
cagagtgcct gttctccctt attaatggag acgatatgtt tacaacgttt 1561
tcaatcatgc aggagaagag ctacttggtt tggctgttta gtcgcattta tttgtattcc
1621 tttataagtc tcttcatata catggttctg agtctcttca ttgcacttat
tactgacaca 1681 tacgatacaa tcaagaatta ccagatcgat ggctttccag
aatcagaact tcacacattt 1741 gtatccgagt gcaaagattt gccaacctct
ggtcgatata gggaacaaga cgagacctcc 1801 tgtttgtcta tgctgtgttg
taatcggtaa aaaagaatcc cagaagaagc actttatcca 1861 tggcctttaa
aaatctgcaa aaaaaaaaaa aaaaaa // Xenopus tropicalis (Silurana
tropicalis) (CAJ83717.1) (GI:89273945) (SEQ ID NO:34)
MENPELIKTCNSLDEHDGPYCCKQCPMTDELLMEDQLRRKLKFFFMNPCEKFRARGRKPWKLCIQILKIA
MVTIQLVLFGLSNEMVVTFKEENTVAFKHLFLKGYKDGHDDTYAIYSQEDVHAHINFTIKRYLELQNISV
GNHAYESNGKGQTGMSLCQHYYKQGSIFPGNETFEIDPQIDTECFHIDPSTLCSNDTPAEYYWSNMTLDF
YRLVSVEIMFKLKAINLQTIRHHELPDCYDFMVIITFDNKAHSGRIKISLDNDVGIQECKDWHVSGSIQK
NTHYMMIFDAAVILVCLSSITLCIRSVVKGIHLQKEYVNFFQHRFARTVSSADRMEFVNGWYIMIIISDV
LSIIGSILKMEIQAKSLTSYDVCSILLGTSTLLVWLGVIRYLGFFKKYNLLILTLRAALPNVIRFCCCAA
MIYLGYCFCGWIVLGPYHVKFRSLNMVSECLFSLINGDDMFTTFSIMQEKSYLVWLFSRIYLYSFISLFI
YMVLSLFIALITDTYDTIKNYQIDGFPESELHTFVSECKDLPTSGRYREQDETSCLSMLCCNR
Homo sapiens Sodium Leak Channel, Non-Selective (NALCN), mRNa
(NM_052867.2) GI:93277089 (SEQ ID NO:35)
gi|93277089|ref|NM_052867.2|Homo sapiens sodium leak channel, non-
selective (NALCN), mRNA
CGCGCTGCCTGAGCTGAGCCGCCGTAGGTGAGGGGCCCGCGTCCCCGCCCGCCCTGGGCGCCGCGCCTGG
CACTGATCCTGCCGGTCGCCCACTGTCGCCGCCGCCGCCGCCCGCGGGCACCATGACAGCTCTGAGCGCT
GGGGTTACAGACTGTGGTTTTGTGCTTGCTCACCAAAGCTAACCTCAGCATGCTCAAAAGGAAGCAGAGT
TCCAGGGTGGAAGCCCAGCCAGTCACTGACTTTGGTCCTGATGAGTCTCTGTCGGATAATGCTGACATCC
TCTGGATTAACAAACCATGGGTTCACTCTTTGCTGCGCATCTGTGCCATCATCAGCGTCATTTCTGTTTG
TATGAATACGCCAATGACCTTCGAGCACTATCCTCCACTTCAGTATGTGACCTTCACTTTGGATACATTA
TTGATGTTTCTCTACACGGCAGAGATGATAGCAAAAATGCACATCCGGGGCATTGTCAAGGGGGATAGTT
CCTATGTGAAAGATCGCTGGTGTGTTTTTGATGGATTTATGGTCTTTTGCCTTTGGGTTTCTTTGGTGCT
ACAGGTGTTTGAAATTGCTGATATAGTTGATCAGATGTCACCTTGGGGCATGTTGCGGATTCCACGGCCA
CTGATTATGATCCGAGCATTCCGGATTTATTTCCGATTTGAACTGCCAAGGACCAGAATTACAAATATTT
TAAAGCGATCGGGAGAACAAATATGGAGTGTTTCCATTTTTCTACTTTTCTTTCTACTTCTTTATGGAAT
TTTAGGAGTTCAGATGTTTGGAACATTTACTTATCACTGTGTTGTAAATGACACAAAGCCAGGGAATGTA
ACCTGGAATAGTTTAGCTATTCCAGACACACACTGCTCACCAGAGCTAGAAGAAGGCTACCAGTGCCCAC
CTGGATTTAAATGCATGGACCTTGAAGATCTGGGACTTAGCAGGCAAGAGCTGGGCTACAGTGGCTTTAA
TGAGATAGGAACTAGTATATTCACCGTCTATGAGGCCGCCTCACAGGAAGGCTGGGTGTTCCTCATGTAC
AGAGCAATTGACAGCTTTCCCCGTTGGCGTTCCTACTTCTATTTCATCACTCTCATTTTCTTCCTCGCCT
GGCTTGTGAAGAACGTGTTTATTGCTGTTATCATTGAAACATTTGCAGAAATCAGAGTACAGTTTCAACA
AATGTGGGGATCGAGAAGCAGCACTACCTCAACAGCCACCACCCAGATGTTTCATGAAGATGCTGCTGGA
GGTTGGCAGCTGGTAGCTGTGGATGTCAACAAGCCCCAGGGACGCGCCCCAGCCTGCCTCCAGAAAATGA
TGCGGTCATCCGTTTTCCACATGTTCATCCTGAGCATGGTGACCGTGGACGTGATCGTGGCGGCTAGCAA
CTACTACAAAGGAGAAAACTTCAGGAGGCAGTACGACGAGTTCTACCTGGCGGAGGTGGCTTTTACAGTA
CTTTTTGATTTGGAAGCACTTCTGAAGATATGGTGTTTGGGATTTACTGGATATATTAGCTCATCTCTCC
ACAAATTCGAACTACTACTCGTAATTGGAACTACTCTTCATGTATACCCAGATCTTTATCATTCACAATT
CACGTACTTTCAGGTTCTCCGAGTAGTTCGGCTGATTAAGATTTCACCTGCATTAGAAGACTTTGTGTAC
AAGATATTTGGTCCTGGAAAAAAGCTTGGGAGTTTGGTTGTATTTACTGCCAGCCTCTTGATTGTTATGT
CAGCAATTAGTTTGCAGATGTTCTGCTTTGTCGAAGAACTGGACAGATTTACTACGTTTCCGAGGGCATT
TATGTCCATGTTCCAGATCCTCACCCAGGAAGGATGGGTGGACGTAATGGACCAAACTCTAAATGCTGTG
GGACATATGTGGGCACCCGTGGTTGCCATCTATTTCATTCTCTATCATCTTTTTGCCACTCTGATCCTCC
TGAGTTTGTTTGTTGCTGTTATTTTGGACAACTTAGAACTTGATGAAGACCTAAAGAAGCTTAAACAATT
AAAGCAAAGTGAAGCAAATGCGGACACCAAAGAAAAGCTCCCTTTACGCCTGCGAATCTTTGAAAAATTT
CCAAACAGACCTCAAATGGTGAAAATCTCAAAGCTTCCTTCAGATTTTACAGTTCCTAAAATCAGGGAGA
GTTTTATGAAGCAGTTTATTGACCGCCAGCAACAGGACACATGTTGCCTCCTGAGAAGCCTCCCGACCAC
CTCTTCCTCCTCCTGCGACCACTCCAAACGCTCAGCAATTGAGGACAACAAATACATCGACCAAAAACTT
CGCAAGTCTGTTTTCAGCATCAGGGCAAGGAACCTTCTGGAAAAGGAGACCGCAGTCACTAAAATCTTAA
GAGCTTGCACCCGACAGCGCATGCTGAGCGGATCATTTGAGGGGCAGCCCGCAAAGGAGAGGTCAATCCT
CAGCGTGCAGCATCATATCCGCCAAGAGCGCAGGTCACTAAGACATGGATCAAACAGCCAGAGGATCAGC
AGGGGAAAATCTCTTGAAACTTTGACTCAAGATCATTCCAATACAGTGAGATATAGAAATGCACAAAGAG
AAGACAGTGAAATAAAGATGATTCAGGAAAAAAAGGAGCAAGCAGAGATGAAAAGGAAAGTGCAAGAAGA
GGAACTCAGAGAGAACCACCCATACTTCGATAAGCCACTGTTCATTGTCGGGCGAGAACACAGGTTCAGA
AACTTTTGCCGGGTGGTGGTCCGAGCACGCTTCAACGCATCTAAAACAGACCCTGTCACAGGAGCTGTGA
AAAATACAAAGTACCATCAACTTTATGATTTGCTGGGATTGGTCACTTACCTGGACTGGGTCATGATCAT
CGTAACCATCTGCTCTTGCATTTCCATGATGTTTGAGTCCCCGTTTCGAAGAGTCATGCATGCACCTACT
TTGCAGATTGCTGAGTATGTGTTTGTGATATTCATGAGCATTGAGCTTAATCTGAAGATTATGGCAGATG
GCTTATTTTTCACTCCAACTGCTGTCATCAGGGACTTCGGTGGAGTAATGGACATATTTATATATCTTGT
GAGCTTGATATTTCTTTGTTGGATGCCTCAAAATGTACCTGCTGAATCGGGAGCTCAGCTTCTAATGGTC
CTTCGGTGCCTGAGACCTCTGCGCATATTCAAACTGGTGCCCCAGATGAGGAAAGTTGTTCGAGAACTTT
TCAGCGGCTTCAAGGAAATTTTTTTGGTCTCCATTCTTTTGCTGACATTAATGCTCGTTTTTGCAAGCTT
TGGAGTTCAGCTTTTTGCTGGAAAACTGGCCAAGTGCAATGATCCCAACATTATTAGAAGGGAAGATTGC
AATGGCATATTCAGAATTAATGTCAGTGTGTCAAAGAACTTAAATTTAAAATTGAGGCCTGGAGAGAAAA
AACCTGGATTTTGGGTGCCCCGTGTTTGGGCGAATCCTCGGAACTTTAATTTCGACAATGTGGGAAACGC
TATGCTGGCGTTGTTTGAAGTTCTCTCCTTGAAAGGCTGGGTGGAAGTGAGAGATGTTATTATTCATCGT
GTGGGGCCGATCCATGGAATCTATATTCATGTTTTTGTATTCCTGGGTTGCATGATTGGACTGACCCTTT
TTGTTGGAGTAGTTATTGCTAATTTCAATGAAAACAAGGGGACGGCTTTGCTGACCGTCGATCAGAGAAG
ATGGGAAGACCTGAAGAGCCGACTGAAGATCGCACAGCCTCTTCATCTTCCGCCTCGCCCGGATAATGAT
GGTTTTAGAGCTAAAATGTATGACATAACCCAGCATCCATTTTTTAAGAGGACAATCGCATTACTCGTCC
TGGCCCAGTCGGTGTTGCTCTCTGTCAAGTGGGACGTCGAGGACCCGGTGACCGTACCTTTGGCAACAAT
GTCAGTTGTTTTCACCTTCATCTTTGTTCTGGAGGTTACCATGAAGATCATAGCAATGTCGCCTGCTGGC
TTCTGGCAAAGCAGAAGAAACCGATACGATCTCCTGGTGACGTCGCTTGGCGTTGTATGGGTGGTGCTTC
ACTTTGCCCTCCTGAATGCATATACTTACATGATGGGCGCTTGTGTGATTGTATTTAGGTTTTTCTCCAT
CTGTGGAAAACATGTAACGCTAAAGATGCTCCTCTTGACAGTGGTCGTCAGCATGTACAAGAGCTTCTTT
ATCATAGTAGGCATGTTTCTCTTGCTGCTGTGTTACGCTTTTGCTGGAGTTGTTTTATTTGGTACTGTGA
AATATGGGGAGAATATTAACAGGCATGCAAATTTTTCTTCGGCTGGAAAAGCTATTACCGTACTGTTCCG
AATTGTCACAGGTGAAGACTGGAACAAGATTATGCATGACTGTATGGTTCAGCCTCCGTTTTGTACTCCA
GATGAATTTACATACTGGGCAACAGACTGTGGAAATTATGCTGGGGCACTTATGTATTTCTGTTCATTTT
ATGTCATCATTGCCTACATCATGCTAAATCTGCTTGTAGCCATAATTGTGGAGAATTTCTCCTTGTTTTA
TTCCACTGAGGAGGACCAGCTTTTAAGTTACAATGATCTTCGCCACTTTCAAATAATATGGAACATGGTG
GATGATAAAAGAGAGGGGGTGATCCCCACGTTCCGCGTCAAGTTCCTGCTGCGGCTACTGCGTGGGAGGC
TGGAGGTGGACCTGGACAAGGACAAGCTCCTGTTTAAGCACATGTGCTACGAAATGGAGAGGCTCCACAA
TGGCGGCGACGTCACCTTCCATGATGTCCTGAGCATGCTTTCATACCGGTCCGTGGACATCCGGAAGAGC
TTGCAGCTGGAGGAACTCCTGGCGAGGGAGCAGCTGGAGTACACCATAGAGGAGGAGGTGGCCAAGCAGA
CCATCCGCATGTGGCTCAAGAAGTGCCTGAAGCGCATCAGAGCTAAACAGCAGCAGTCGTGCAGTATCAT
CCACAGCCTGAGAGAGAGTCAGCAGCAAGAGCTGAGCCGGTTTCTGAACCCGCCCAGCATCGAGACCACC
CAGCCCAGTGAGGACACGAATGCCAACAGTCAGGACAACAGCATGCAACCTGAGACAAGCAGCCAGCAGC
AGCTCCTGAGCCCCACGCTGTCGGATCGAGGAGGAAGTCGGCAAGATGCAGCCGACGCAGGGAAACCCCA
GAGGAAATTTGGGCAGTGGCGTCTGCCCTCAGCCCCAAAACCAATAAGCCATTCAGTGTCCTCAGTCAAC
TTACGGTTTGGAGGAAGGACAACCATGAAATCTGTCGTGTGCAAAATGAACCCCATGACTGACGCGGCTT
CCTGCGGTTCTGAAGTTAAGAAGTGGTGGACCCGGCAGCTGACTGTGGAGAGCGACGAAAGTGGGGATGA
CCTTCTGGATATTTAGGTGGATGTCAATGTAGATGAATTTCTAGTGGTGGAAACCGTTTTCTAATAATGT
CCTTGATTGTCCAGTGAGCAATCTGTAATTGATCTATAACTGAATTCCAGCTTGTCACAAGATGTTTATA
AATTGATTTTCATCCTGCCACAGAAAGGCATAAGCTGCATGTATGATGGGTTACTATCAATCATTGCTCA
AAAAAATTTTTGTATAATGACAGTACTGATAATATTAGAAATGATACCGCAAGCAAATGTATATCACTTA
AAAATGTCATATATTCTGTCTGCGTAAACTAAGGTATATATTCATATGTGCTCTAATGCAGTATTATCAC
CGCCCCGCAAAAGAGTGCTAAGCCCAAAGTGGCTGATATTTAGGGTACAGGGGTTATAGCTTTAGTTCAC
ATCTTTCCCATTTCCACTAGAAATATTTCTCTTGAGAGAATTTATTATTTATGATTGATCTGAAAAGGTC
AGCACTGAACTTATGCTAAAATGATAGTAGTTTTACAAACTACAGATTCTGAATTTTAAAAAGTATCTTC
TTTTTCTCGTGTTATATTTTTAAATATACACAAGACATTTGGTGACCAGAACAAGTTGATTTCTGTCCTC
AGTTATGTTAATGAAACTGTTGCCTCCTTCTAAGAAAATTGTGTGTGCAAGCACCAGGCAAAGAAATGGA
CTCAGGATGCTTAGCGGTTTAAAACAAACCTGTAGATAAATCACTTGAGTGACATAGTTGCGCAAAGATG
TTAAGTTTCTTAAGAAACCTTTAATAACTGAGTTTAGCAAAAAGAATAAAACTATATAGCTCAATTTATT
TAAAAAAATCTTTGCATGTGTGATGTTATCATTGGCTTCATTTCTTACCCAAGGTATGTCTGTTTTGCCA
TAAATCAGCAGAGTCATTTCATTCTGGGTGATCCTAACACACCATTGCTACGTTAGATTTGAAATGACAT
CTCTGTTAAAAGAATCTTCTATGGAAATAATGGTGCCCTGCAAAATCTTCCTTTGAACTCACAGGTTAGG
GATCACACAACTTACTTAATCGTTTTTTGTTTTTGTTTTTTTTCCTTATATGTCAATGGCCCATGTCCTC
CGGGAAAATTAGAAAAGCAAAATGATTACAAAGTGCTGTTAGATTTCTTGTGCTGGGCCAGCCAAGTAGA
AGTGGACTTGACTTGGACCTTTAACTATTTTATTACAGATTGGACATTTGCTGTTCAGATGTTTTTTAAC
AGAGGGATTATCTCAGAATCCTGTGACCTCCAGGTTGTTTTATAATCTATTTTTCTCTATTTAACATTCC
TCAGATAGATAGGCAAATAGGACATTCCTTCTGTGTCACAGAAGTATCGTGGTAGTGGCAGTCTACAGTT
TATATGATTCATTGTAACTATGAGATAAAGAACAACCAGTCATGTGGCCAAAAGGATTAGATTTGATTTG
ATGTTCACTTGGAGTTTACTTTTTGTACATACAAGATAAAATAAATATTGGATTTGTAAAAT Homo
sapiens Sodium Leak Channel, Non-Selective (NALCN), Polypeptide
(NP_443099.1) GI:24119274 (SEQ ID NO:36)
MLKRKQSSRVEAQPVTDFGPDESLSDNADILWINKPWVHSLLRICAIISVISVCMNTPMTFEHYPPLQYV
TFTLDTLLMFLYTAEMIAKMHIRGIVKGDSSYVKDRWCVFDGFMVFCLWVSLVLQVFEIADIVDQMSPWG
MLRIPRPLIMIRAFRIYFRFELPRTRITNILKRSGEQIWSVSIFLLFFLLLYGILGVQMFGTFTYHCVVN
DTKPGNVTWNSLAIPDTHCSPELEEGYQCPPGFKCMDLEDLGLSRQELGYSGFNEIGTSIFTVYEAASQE
GWVFLMYRAIDSFPRWRSYFYFITLIFFLAWLVKNVFIAVIIETFAEIRVQFQQMWGSRSSTTSTATTQM
FHEDAAGGWQLVAVDVNKPQGRAPACLQKMMRSSVFHMFILSMVTVDVIVAASNYYKGENFRRQYDEFYL
AEVAFTVLFDLEALLKIWCLGFTGYISSSLHKFELLLVIGTTLHVYPDLYHSQFTYFQVLRVVRLIKISP
ALEDFVYKIFGPGKKLGSLVVFTASLLIVMSAISLQMFCFVEELDRFTTFPRAFMSMFQILTQEGWVDVM
DQTLNAVGHMWAPVVAIYFILYHLFATLILLSLFVAVILDNLELDEDLKKLKQLKQSEANADTKEKLPLR
LRIFEKFPNRPQMVKISKLPSDFTVPKIRESFMKQFIDRQQQDTCCLLRSLPTTSSSSCDHSKRSAIEDN
KYIDQKLRKSVFSIRARNLLEKETAVTKILRACTRQRMLSGSFEGQPAKERSILSVQHHIRQERRSLRHG
SNSQRISRGKSLETLTQDHSNTVRYRNAQREDSEIKMIQEKKEQAEMKRKVQEEELRENHPYFDKPLFIV
GREHRFRNFCRVVVRARFNASKTDPVTGAVKNTKYHQLYDLLGLVTYLDWVMIIVTICSCISMMFESPFR
RVMHAPTLQIAEYVFVIFMSIELNLKIMADGLFFTPTAVIRDFGGVMDIFIYLVSLIFLCWMPQNVPAES
GAQLLMVLRCLRPLRIFKLVPQMRKVVRELFSGFKEIFLVSILLLTLMLVFASFGVQLFAGKLAKCNDPN
IIRREDCNGIFRINVSVSKNLNLKLRPGEKKPGFWVPRVWANPRNFNFDNVGNAMLALFEVLSLKGWVEV
RDVIIHRVGPIHGIYIHVFVFLGCMIGLTLFVGVVIANFNENKGTALLTVDQRRWEDLKSRLKIAQPLHL
PPRPDNDGFRAKMYDITQHPFFKRTIALLVLAQSVLLSVKWDVEDPVTVPLATMSVVFTFIFVLEVTMKI
IAMSPAGFWQSRRNRYDLLVTSLGVVWVVLHFALLNAYTYMMGACVIVFRFFSICGKHVTLKMLLLTVVV
SMYKSFFIIVGMFLLLLCYAFAGVVLFGTVKYGENINRHANFSSAGKAITVLFRIVTGEDWNKIMHDCMV
QPPFCTPDEFTYWATDCGNYAGALMYFCSFYVIIAYIMLNLLVAIIVENFSLFYSTEEDQLLSYNDLRHF
QIIWNMVDDKREGVIPTFRVKFLLRLLRGRLEVDLDKDKLLFKHMCYEMERLHNGGDVTFHDVLSMLSYR
SVDIRKSLQLEELLAREQLEYTIEEEVAKQTIRMWLKKCLKRIRAKQQQSCSIIHSLRESQQQELSRFLN
PPSIETTQPSEDTNANSQDNSMQPETSSQQQLLSPTLSDRGGSRQDAADAGKPQRKFGQWRLPSAPKPIS
HSVSSVNLRFGGRTTMKSVVCKMNPMTDAASCGSEVKKWWTRQLTVESDESGDDLLDI Homo
sapiens Sodium/Potassium Transporting ATPase Interacting 3 (NKAIN3)
(NP_173688.2) GI:14538007 (SEQ ID NO:3) 1 agccgcgagc ggcggccgcg
gggccgagga gcctgggccg ggccgggcgg ggactactcc 61 ggagtcagga
ggcagcagcg gcggaggacg aggatctctg gcagtcagcg ccgctcggac 121
gccgccggca ccatgggctg ctgcaccgga cgctgctcgc tcatctgcct ctgcgcgctg
181 cagttggtct cagcattaga gaggcagatc tttgacttcc ttggtttcca
gtgggcgcct 241 attcttggaa attttctaca cataatagtt gtcatattgg
gtttgtttgg gaccattcag 301 tacagacctc gatacataat ggtgtataca
gtgtggactg ccctctgggt cacctggaat 361 gtgttcatta tctgctttta
tttggaagta ggtggactct caaaggacac cgatctaatg 421 acattcaata
tctctgtaca tcggtcatgg tggagagaac atgggcctgg ttgtgtcaga 481
agagtgctgc ctccctcagc ccatggcatg atggacgatt acacgtacgt ctctgtcaca
541 ggctgcatcg ttgacttcca gtacctggag gtcatccaca gtgctgtcca
aatactactc 601 tctttggtgg gttttgtgta tgcctgttat gtgatcagta
tttccatgga agaagaagac 661 acatattcat gtgatctgca agtatgcaaa
catcttttta tccagatgct gcaaattatt 721 gaataagcaa gaattagtaa
gatattatca ccaaattgtc acatcagtca agcctcatgt 781 gcttcctaag
aactgaggtg atgcattatt ttagagtgtc attctaaacc ccagattcaa 841
catcttccta atctttctag tgcagtctaa tatataaatt ttatgaaaag cataggtttt
901 tttttaacca gcagtgctct ttgagaattt acattgattc ctaaagattg
ccattgcttt 961 gtataaaatg ttataaatta tcttagcatc ttacctggaa
tttccactaa attcaccaat 1021 ttatgatttg tgaaatctga ttttactttt
tgaaaatttt catgtgaatt tcccattttc 1081 agtgttgtag cacctctctc
ttcctctaag atcctccaag ctcatcaaaa gccatgatct 1141 tattatacca
gcagttttat ttattcaatc tttcaacaag tagttattga acttctataa 1201
tgtgccaggc tctggagctc gccttacacc aaacagacac aatcgatcca ttcgaagtgt
1261 cgtaattaca cattgaggga ccaactagac cttttctcat tgtaaacttg
gagcaaaagt 1321 aaattcatta aaataaattt acattatagt gccacaaaaa
aatgaacaga accagaaagc 1381 attttttaca aaaattaaca gaacagtgtg
atagagggga aaaggatgtg agatcatggt 1441 gccctacctt caatagggtg
gccagaaaac acctctctga agaagcagca tttgagctga 1501 gacctgaaga
acgaggagtc agtgatgcag agaacctcag gagatgcctt ccaatctgag 1561 aaaaga
Homo sapiens Sodium/Potassium Transporting ATPase Interacting 3
(NKAIN3) Polypeptide (NP_775959.1) GI:27735000 (SEQ ID NO:38)
MGCCTGRCSLICLCALQLVSALERQIFDFLGFQWAPILGNFLHIIVVILGLFGTIQYRPRYIMVYTVWTA
LWVTWNVFIICFYLEVGGLSKDTDLMTFNISVHRSWWREHGPGCVRRVLPPSAHGMMDDYTYVSVTGCIV
DFQYLEVIHSAVQILLSLVGFVYACYVISISMEEEDTYSCDLQVCKHLFIQMLQIIE >Homo
sapiens Mucolipin 3, mRNA gi|38174237|gb|BC060765.1| (SEQ ID NO:39)
TGGAGTCGCTCGCTGACTCGCCCTGCGCCCTCGCCGCGGACACCGGAGCTGCGGCCGCTCCCCGCTGTCC
CCCAGCTTACTCCAATCAAGCCTCTGCCCGCCAGGAACAGGTAACCTGTGTGTGTCCGTTTGCTCCTTCT
AAGAGCATGCCTGATAGATACTTCGGTAGCCTCTCCGGATGGCCCCTTCGTCGGGTAGCCTCTCCTGATG
GGGTCCTTCGCCCACCCTGCCTCCCGCGCCGGCGCTCCGGGTGAATGTCAAGGGTGGCTGGCTGCGAATA
CCTCCTTCAGCTGCTGGGGTTCCCGACAGTTTGCAGTTTTTAAAAGTGCACCCTCGGAAGGGCTTTTCAG
ACTGGGTAAACCTGACTTTTCCAAGAGATGGCAGATCCTGAGGTAGTTGTGAGTAGCTGCAGCTCTCATG
AAGAGGAAAATCGCTGCAATTTTAACCAGCAAACATCTCCATCTGAGGAGCTTCTATTAGAAGACCAGAT
GAGGCGAAAACTCAAATTTTTTTTCATGAATCCCTGTGAGAAGTTCTGGGCTCGAGGTAGAAAACCATGG
AAACTTGCCATACAAATTCTAAAAATTGCAATGGTGACTATCCAGCTGGTCTTATTTGGGCTAAGTAACC
AGATGGTGGTAGCTTTCAAGGAAGAGAATACTATAGCATTCAAACACCTTTTCCTAAAAGGATATATGGA
CCGAATGGATGACACATATGCAGTGTACACACAAAGTGACGTGTATGATCAGTTAATCTTCGCAGTAAAC
CAGTACTTGCAGCTATACAATGTCTCCGTTGGGAATCATGCTTATGAGAACAAAGGTACCAAGCAATCTG
CTATGGCAATCTGTCAGCACTTATACAAGCGAGGAAACATCTACCCTGGAAATGATACCTTTGACATCGA
TCCAGAAATTGAAACTGAGTGTTTCTTTGTGGAGCCAGATGAACCTTTTCACATTGGGACACCAGCAGAA
AATAAACTGAACTTAACACTGGACTTCCACAGACTCCTAACAGTGGAGCTTCAGTTTAAACTGAAAGCCA
TTAATCTGCAGACAGTTCGTCATCAAGAACTCCCTGACTGTTATGACTTTACTCTGACTATAACATTTGA
CAACAAGGCCCATAGTGGAAGAATTAAAATAAGTTTAGATAATGACATTTCCATCAGAGAATGTAAAGAC
TGGCATGTATCTGGATCAATTCAGAAGAACACTCATTACATGATGATCTTTGATGCCTTTGTCATTCTGA
CTTGCTTGGTTTCATTAATCCTCTGCATTAGATCTGTGATTAGAGGACTTTCAGCTTCAGCAGGTAGGGA
ACGTTGCTTTCTAGGAATGCTACTGACATTTTGATTGACAGAGACATTCACTGTGCCTCCCCTTTTCCCT
AAAGGAGTTTGTCAATTTTTTCCTCCTCCATTATAAGAAGGAAGTTTCTGTTTCTGATCAAATGGAATTT
GTCAATGGATGGTACATTATGATTATTATTAGTGACATATTGACAATCATTGGATCAATTCTAAAAATGG
AAATCCAAGCTAAGGTAATTTTTTTCCTAATCATGCTATTGTTAGTGTCAGATTTGCACTAATGGTAATG
TATTTTTCCAGAATGTAAGAATTTTCAGAATGAATTGTTTCTTCCAAACTGTATATCAAGTAGACTTGAA
ATTGGTAATGGTAATTTTCTTAAATCTAGTCAGGAGGTCTCTTAGGCAGAGTTTTTCAAAGTGTGATCCA
CAAACCATTGCATCAGAATCATTGGGTGCCTGGTAAAGTGTACCATGTTAGACCTACTGAATTCAGACTC
TTCGGCGGGGCCTGTGAATTCTTACACACACCAAAATTCATACACAACCAAGGTAACTAAGGTAAGAGTT
TTTTTTTTTTTTTAATCTTACAAGAAATGCTCGAATCTTTAACAAAAATGAGTGGGGCTATAGGGGAAAG
TGAGGTCAAGGCACTATGGTGTGCATGCTTGCATTTGTTTCCTCCGTCCATTCAAAGTGAGAATGCTCCC
ATTTTCTTACTTTACCATTGATGTGCTACAAGCTTATTTATTTTAAGACTAACCTAGCCTAAAAATCAAC
TGTCCCCACAAAATAAAAATCACATTAAAAAAACTAATAGTGTTCAGACTAATCTTGCTCAAACTTATGT
TTCCCTAGTCTTGATGCAACTGATTGAGTCACCTGGGGAGTTGGTTATAAACCTGGGCAGAGACCCCAAA
TGCAATGGCTCAGAGAAGATAGGAGCTTATTTCTGTCTTATGCAATAGTCAGAATGGGTTTTACAGACTG
GTGAGTAGCTCAACATCTCACAGTCATTCAGGCACCCATGTTCCTCCCATTTTGTTTCTCTGCCACCCCT
TAAGGACTTGCCCTGACTGCATGATTATTGCCGTGTTGCCTCAAACAGGTTGCAGCTTATGGGAAGCAAA
AACACGGTATGGTAGAAGCTCTCCCATAGACTGATGGCTTGGCTCAAGAGTGGCCGACTTTATTTCTGTA
CATATCCCACTGGATAGAATTTAGTCAATCCTAACTGCAGAGGGAGCCAGGGAACACAGCCCAGGCATGT
GCCTAGGAAGGGGAGAATGGGTTTAGGTTGACACTTAGCAGCTGCCACTATATGTGGCTATAGTATGTAT
CATTGGAATAGATGTTTAACTTTAGGGACAAATAAAAAACCAAAACAAAAAAAGGAGTAAGGGGAGAGAT
TTGCAGCAAATCTTTATTTTTACCAACCTCAACTATCATTAATTTCAGTGAACCCTAAATGGTATCCAAC
AAAATATCTTTCTAGACCATTCACCGTCTCTGCCTCATAGATGATCATATCATGTTTTCTTCTCTTCTGA
AACCTCTAATACCCTTGTCCTATCCTCATTCTAAGCTGATGACCTTACTTCCTATTTCACAAAAATAATA
GAAAAAAAAAAAAAAAA >>Homo sapiens Mucolipin 3, cDNA
gi|21752911|dbj|AK093948.1 Homo sapiens cDNA FLJ36629 (SEQ ID
NO:40)
ATAGCCTTTCAAATTTCGGTTAATGGTAACTCTCATCAGTTACTCAAGCCAAAAATCTTGGATTCATCAC
AGACTCCTCTCTTTCACTAATCTCTTCTTCCCCACATCTAATCCAAGAGGAAATCCTATTGTTCTACCTT
CATTGGCCAGGCACTGTGACTCATATAATCCCAGCACATTGGGAGGCTGAGGCGGGAGGATGGCTTGAGC
CCAGGAGTTCCAGACCAGCCTGGGAAACATAGTGAGGCCCCATCTCTACAAAAAATTAAAAAAATTAGCA
GTCAAGGTGGCATGTGTCTGTAATCCAAGCTACTTGGGGGGCTGAGGTGGGAGGATTTCTTGAGCCCGGG
AAGTCGAGGCTGCATTGAGCCACGGTTGTGCCACGGCACTCCAGCCTGGGTGACAGAGTGAGACCCCTGT
TTCCAAAAAAAAAAAAAAAAAAAAAGAATAAAAATCACCTGGTCAACTCCACTATTACCAGCCTGGTCCA
AGCTACTATCTCTCATTTATATTATTGCAATAGCCTCCTCACTCCTCCAACAACCTGCTGTCAACCACAG
CAGCCAACATCTGATCATATCACTTCTGTTTGTGGTTCTCAAATCTCCCCAATTGAGTTACAGTAAAAGA
CAAACTTGGTGAGTGCCACCTTATCTCTATAACTGTATACCCTTTCTATTGCTCACTCCAGCCAGATGCA
ATATCCTTGCCAAGCACCCTCCTGTCTCAGGGCCTTTGCACTTGCCAGTCCCTGTGCCTGGAAGGCTTCT
CCCCTAGATTTTTGCATGACTTCTCCCTCCCTCCCTTCAGATCTTTGCTCAAATGCCTTCTTTTTAGTGT
ATGTAAAATGACAAACCCATACCCATTCCTTATCCCCTCCTCTGAATTTTCTCTTCAGCAATTATCAGCA
GCAAGTGTCCCAAAGTTTCTATTAACTTATTTCTGTTGTCTCTTTCTTCCCTCCACTAGAATGTAAGCTT
TATGAGAGCAGAGACTTTTGTTTGTTCACTGCTTTATCCTTAGCACCTAAAACAGTGCCTTACTCATAGT
TACCTCAATATTTATTGCCAAATGAATTTCTGCTTTATAATCTGATTATATTTTTCCACTCTCTCTTAGA
GTCTAACTAGTTATGATGTCTGTAGCATACTTCTTGGGACTTCTACCATGCTTGTGTGGCTTGGAGTCAT
CCGATACCTCGGTTTCTTTGCAAAGTACAACCTCCTCATTTTGACCCTTCAGGCAGCGCTGCCCAATGTC
ATCAGGTTCTGCTGCTGTGCAGCTATGATTTACTTAGGTTACTGCTTCTGTGGATGGATCGTGCTGGGGC
CTTACCATGACAAGTTTCGTTCTCTGAACATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGA
TATGTTTGCCACGTTTGCAAAAATGCAGCAAAAAAGTTACTTAGTCTGGCTGTTTAGTAGAATTTACCTC
TACTCATTCATCAGCCTCTTTATATATATGATTTTAAGTCTTTTCATTGCACTGATCACTGATACATACG
AAACAATTAAGCAATACCAACAAGATGGCTTCCCAGAGACTGAACTTCGTACATTTATATCAGAATGCAA
AGATCTACCCAACTCTGGAAAATACAGATTAGAAGATGACCCTCCAGTAACTTTATTCTGCTGTTGTAAA
AAGTAGCTATCAGGTTTATCTGTACTTTAGAGGAAAATATAATGTGTAGCTGAGTTGGAACACTGTGGAT
ATTCTGAGATCAGATGTAGTATGTTTGAAGACTGTTATTTTGAGCTAATTGAGACCTATAATTCACCAAT
AACTGTTTATATTTTTAAAAGCAATATTTAATGTCTTTGCAACTTTATGCTGGGATTGTTTTTAAAAAAA
ACTTTAATGAGGAAAGCTATTGGATTATTATTATTTCTTGTTTATTTTGCCATGGCTTTAGAATGTATTC
TGTATGCCTCTCTTTTGCTCTGATACTGTTGCTCCTGCTATTCTGATTGTGCAGACTGTATAATTAGTGG
AAAACAATCCTTGGTCTGACTGTGACTTTGGACAACTCAGTAACCCTGGCTTGGACCACTCTCAGGAGTC
CATCCTTGAGAGAGTGGGTGTAGTTACCATTTATACAGTAATCATTGCATTTTAAAATCTTCTCTTGAAA
GGAAGAATAAGAGTGCACCAGAATAAGAGCGCACCAGAATAAGAGCACACCAGCTAACAATGTGATACGG
CCATATGTCACTTAAGGATGGAGATATGTTCTGAGAAATGTGTCATTAGGCGATTTTGTCATTAAACATC
ATAGCATGTACTTCCACAAACCTAGATGGTATAGCCTACTACACACCTAGGCTATTTGGTATAGCCTGTT
GGTCCTGGGGTACAAATCTGTACAACATGTTACTGTATTGAATACAGTAGGCAATTGTAACTCAATGGTA
AGTATCTAAACATAGAAAAGGGACAGTAAAAATATGGTTTTATAATCTTCTGGGACCACCATTGTATATG
CGGTACATCATTGACCAAAACATCGTTATCCAGCATATGACTGTATTTGGTTATGAAAGCCAACTGTTAC
TTGATTCTGCTTTTAGTTCTTAAGAGGATCAGGCTTTTAAATACTCATTTACAAGCTTTCTATCCTCCTT
CAGTGTTAAAGTAGAAAGTAAAAAGAGTATCTTATACATGCATGAAATTAAAGCATATACCAAATGC
REFERENCES
[0588] All the references cited in this application are
incorporated by reference in their entirety herein.
Sequence CWU 1
1
4011659DNAHomo sapiens 1atggcagatc ctgaggtagt tgtgagtagc tgcagctctc
atgaagagga aaatcgctgc 60aattttaacc agcaaacatc tccatctgag gagcttctat
tagaagacca gatgaggcga 120aaactcaaat tttttttcat gaatccctgt
gagaagttct gggctcgagg tagaaaacca 180tggaaacttg ccatacaaat
tctaaaaatt gcaatggtga ctatccagct ggtcttattt 240gggctaagta
accagatggt ggtagctttc aaggaagaga atactatagc attcaaacac
300cttttcctaa aaggatatat ggaccgaatg gatgacacat atgcagtgta
cacacaaagt 360gacgtgtatg atcagttaat cttcgcagta aaccagtact
tgcagctata caatgtctcc 420gttgggaatc atgcttatga gaacaaaggt
accaagcaat ctgctatggc aatctgtcag 480cacttctaca agcgaggaaa
catctaccct ggaaatgata cctttgacat cgatccagaa 540attgaaactg
agtgtttctt tgtggagcca gatgaacctt ttcacattgg gacaccagca
600gaaaataaac tgaacttaac actggacttc cacagactcc taacagtgga
gcttcagttt 660aaactgaaag ccattaatct gcagacagtt cgtcatcaag
aactccctga ctgttatgac 720tttactctga ctataacatt tgacaacaag
gcccatagtg gaagaattaa aataagttta 780gataatgaca tttccatcag
agaatgtaaa gactggcatg tatctggatc aattcagaag 840aacactcatt
acatgatgat ctttgatgcc tttgtcattc tgacttgctt ggtttcatta
900atcctctgca ttagatctgt gattagagga cttcagcttc agcaggagtt
tgtcaatttt 960ttcctcctcc attataagaa ggaagtttct gtttctgatc
aaatggaatt tgtcaatgga 1020tggtacatta tgattattat tagtgacata
ttgacaatca ttggatcaat tctaaaaatg 1080gaaatccaag ctaagagtct
aactagttat gatgtctgta gcatacttct tgggacttct 1140accatgctcg
tgtggcttgg agtcatccga tacctcggtt tctttgcaaa gtacaacctc
1200ctcattttga cccttcaggc agcgctgccc aatgtcatca ggttctgctg
ctgtgcagct 1260atgatttact taggttactg cttctgtgga tggatcgtgc
tggggcctta ccatgacaag 1320tttcgttctc tgaacatggt ctctgagtgc
cttttctctc tgataaatgg agatgatatg 1380tttgccacgt ttgcaaaaat
gcagcaaaaa agttacttag tctggctgtt tagtagaatt 1440tacctctact
cattcatcag cctctttata tatatgattt taagtctttt cattgcactg
1500atcactgata catacgaaac aattaagcaa taccaacaag atggcttccc
agagactgaa 1560cttcgtacat ttatatcaga atgcaaagat ctacccaact
ctggaaaata cagattagaa 1620gatgaccctc cagtatcttt attctgctgt
tgtaaaaag 16592553PRTHomo sapiens 2Met Ala Asp Pro Glu Val Val Val
Ser Ser Cys Ser Ser His Glu Glu1 5 10 15Glu Asn Arg Cys Asn Phe Asn
Gln Gln Thr Ser Pro Ser Glu Glu Leu 20 25 30Leu Leu Glu Asp Gln Met
Arg Arg Lys Leu Lys Phe Phe Phe Met Asn 35 40 45Pro Cys Glu Lys Phe
Trp Ala Arg Gly Arg Lys Pro Trp Lys Leu Ala 50 55 60Ile Gln Ile Leu
Lys Ile Ala Met Val Thr Ile Gln Leu Val Leu Phe65 70 75 80Gly Leu
Ser Asn Gln Met Val Val Ala Phe Lys Glu Glu Asn Thr Ile 85 90 95Ala
Phe Lys His Leu Phe Leu Lys Gly Tyr Met Asp Arg Met Asp Asp 100 105
110Thr Tyr Ala Val Tyr Thr Gln Ser Asp Val Tyr Asp Gln Leu Ile Phe
115 120 125Ala Val Asn Gln Tyr Leu Gln Leu Tyr Asn Val Ser Val Gly
Asn His 130 135 140Ala Tyr Glu Asn Lys Gly Thr Lys Gln Ser Ala Met
Ala Ile Cys Gln145 150 155 160His Phe Tyr Lys Arg Gly Asn Ile Tyr
Pro Gly Asn Asp Thr Phe Asp 165 170 175Ile Asp Pro Glu Ile Glu Thr
Glu Cys Phe Phe Val Glu Pro Asp Glu 180 185 190Pro Phe His Ile Gly
Thr Pro Ala Glu Asn Lys Leu Asn Leu Thr Leu 195 200 205Asp Phe His
Arg Leu Leu Thr Val Glu Leu Gln Phe Lys Leu Lys Ala 210 215 220Ile
Asn Leu Gln Thr Val Arg His Gln Glu Leu Pro Asp Cys Tyr Asp225 230
235 240Phe Thr Leu Thr Ile Thr Phe Asp Asn Lys Ala His Ser Gly Arg
Ile 245 250 255Lys Ile Ser Leu Asp Asn Asp Ile Ser Ile Arg Glu Cys
Lys Asp Trp 260 265 270His Val Ser Gly Ser Ile Gln Lys Asn Thr His
Tyr Met Met Ile Phe 275 280 285Asp Ala Phe Val Ile Leu Thr Cys Leu
Val Ser Leu Ile Leu Cys Ile 290 295 300Arg Ser Val Ile Arg Gly Leu
Gln Leu Gln Gln Glu Phe Val Asn Phe305 310 315 320Phe Leu Leu His
Tyr Lys Lys Glu Val Ser Val Ser Asp Gln Met Glu 325 330 335Phe Val
Asn Gly Trp Tyr Ile Met Ile Ile Ile Ser Asp Ile Leu Thr 340 345
350Ile Ile Gly Ser Ile Leu Lys Met Glu Ile Gln Ala Lys Ser Leu Thr
355 360 365Ser Tyr Asp Val Cys Ser Ile Leu Leu Gly Thr Ser Thr Met
Leu Val 370 375 380Trp Leu Gly Val Ile Arg Tyr Leu Gly Phe Phe Ala
Lys Tyr Asn Leu385 390 395 400Leu Ile Leu Thr Leu Gln Ala Ala Leu
Pro Asn Val Ile Arg Phe Cys 405 410 415Cys Cys Ala Ala Met Ile Tyr
Leu Gly Tyr Cys Phe Cys Gly Trp Ile 420 425 430Val Leu Gly Pro Tyr
His Asp Lys Phe Arg Ser Leu Asn Met Val Ser 435 440 445Glu Cys Leu
Phe Ser Leu Ile Asn Gly Asp Asp Met Phe Ala Thr Phe 450 455 460Ala
Lys Met Gln Gln Lys Ser Tyr Leu Val Trp Leu Phe Ser Arg Ile465 470
475 480Tyr Leu Tyr Ser Phe Ile Ser Leu Phe Ile Tyr Met Ile Leu Ser
Leu 485 490 495Phe Ile Ala Leu Ile Thr Asp Thr Tyr Glu Thr Ile Lys
Gln Tyr Gln 500 505 510Gln Asp Gly Phe Pro Glu Thr Glu Leu Arg Thr
Phe Ile Ser Glu Cys 515 520 525Lys Asp Leu Pro Asn Ser Gly Lys Tyr
Arg Leu Glu Asp Asp Pro Pro 530 535 540Val Ser Leu Phe Cys Cys Cys
Lys Lys545 55031659DNAHomo sapiens 3atggcagatc ctgaggtagt
tgtgagtagc tgcagctctc atgaagagga aaatcgctgc 60aattttaacc agcaaacatc
tccatctgag gagcttctat tagaagacca gatgaggcga 120aaactcaaat
tttttttcat gaatccctgt gagaagttct gggctcgagg tagaaaacca
180tggaaacttg ccatacaaat tctaaaaatt gcaatggtga ctatccagct
ggtcttattt 240gggctaagta accagatggt ggtagctttc aaggaagaga
atactatagc attcaaacac 300cttttcctaa aaggatatat ggaccgaatg
gatgacacat atgcagtgta cacacaaagt 360gacgtgtatg atcagttaat
cttcgcagta aaccagtact tgcagctata caatgtctcc 420gttgggaatc
atgcttatga gaacaaaggt accaagcaat ctgctatggc aatctgtcag
480cacttctaca agcgaggaaa catctaccct ggaaatgata cctttgacat
cgatccagaa 540attgaaactg agtgtttctt tgtggagcca gatgaacctt
ttcacattgg gacaccagca 600gaaaataaac tgaacttaac actggacttc
cacagactcc taacagtgga gcttcagttt 660aaactgaaag ccattaatct
gcagacagtt cgtcatcaag aactccctga ctgttatgac 720tttactctga
ctataacatt tgacaacaag gcccatagtg gaagaattaa aataagttta
780gataatgaca tttccatcag agaatgtaaa gactggcatg tatctggatc
aattcagaag 840aacactcatt acatgatgat ctttgatgcc tttgtcattc
tgacttgctt ggtttcatta 900atcctctgca ttagatctgt gattagagga
cttcagcttc agcaggagtt tgtcaatttt 960ttcctcctcc attataagaa
ggaagtttct gtttctgatc aaatggaatt tgtcaatgga 1020tggtacatta
tgattattat tagtgacata ttgacaatca ttggatcaat tctaaaaatg
1080gaaatccaag ctaagagtct aactagttat gatgtctgta gcatacttct
tgggacttct 1140accatgctcg tgtggcttgg agtcatccga tacctcggtt
tctttgcaaa gtacaacctc 1200ctcattttga cccttcaggc agcgctgccc
aatgtcatca ggttctgctg ctgtccagct 1260atgatttact taggttactg
cttctgtgga tggatcgtgc tggggcctta ccatgacaag 1320tttcgttctc
tgaacatggt ctctgagtgc cttttctctc tgataaatgg agatgatatg
1380tttgccacgt ttgcaaaaat gcagcaaaaa agttacttag tctggctgtt
tagtagaatt 1440tacctctact cattcatcag cctctttata tatatgattt
taagtctttt cattgcactg 1500atcactgata catacgaaac aattaagcaa
taccaacaag atggcttccc agagactgaa 1560cttcgtacat ttatatcaga
atgcaaagat ctacccaact ctggaaaata cagattagaa 1620gatgaccctc
cagtatcttt attctgctgt tgtaaaaag 16594553PRTHomo sapiens 4Met Ala
Asp Pro Glu Val Val Val Ser Ser Cys Ser Ser His Glu Glu1 5 10 15Glu
Asn Arg Cys Asn Phe Asn Gln Gln Thr Ser Pro Ser Glu Glu Leu 20 25
30Leu Leu Glu Asp Gln Met Arg Arg Lys Leu Lys Phe Phe Phe Met Asn
35 40 45Pro Cys Glu Lys Phe Trp Ala Arg Gly Arg Lys Pro Trp Lys Leu
Ala 50 55 60Ile Gln Ile Leu Lys Ile Ala Met Val Thr Ile Gln Leu Val
Leu Phe65 70 75 80Gly Leu Ser Asn Gln Met Val Val Ala Phe Lys Glu
Glu Asn Thr Ile 85 90 95Ala Phe Lys His Leu Phe Leu Lys Gly Tyr Met
Asp Arg Met Asp Asp 100 105 110Thr Tyr Ala Val Tyr Thr Gln Ser Asp
Val Tyr Asp Gln Leu Ile Phe 115 120 125Ala Val Asn Gln Tyr Leu Gln
Leu Tyr Asn Val Ser Val Gly Asn His 130 135 140Ala Tyr Glu Asn Lys
Gly Thr Lys Gln Ser Ala Met Ala Ile Cys Gln145 150 155 160His Phe
Tyr Lys Arg Gly Asn Ile Tyr Pro Gly Asn Asp Thr Phe Asp 165 170
175Ile Asp Pro Glu Ile Glu Thr Glu Cys Phe Phe Val Glu Pro Asp Glu
180 185 190Pro Phe His Ile Gly Thr Pro Ala Glu Asn Lys Leu Asn Leu
Thr Leu 195 200 205Asp Phe His Arg Leu Leu Thr Val Glu Leu Gln Phe
Lys Leu Lys Ala 210 215 220Ile Asn Leu Gln Thr Val Arg His Gln Glu
Leu Pro Asp Cys Tyr Asp225 230 235 240Phe Thr Leu Thr Ile Thr Phe
Asp Asn Lys Ala His Ser Gly Arg Ile 245 250 255Lys Ile Ser Leu Asp
Asn Asp Ile Ser Ile Arg Glu Cys Lys Asp Trp 260 265 270His Val Ser
Gly Ser Ile Gln Lys Asn Thr His Tyr Met Met Ile Phe 275 280 285Asp
Ala Phe Val Ile Leu Thr Cys Leu Val Ser Leu Ile Leu Cys Ile 290 295
300Arg Ser Val Ile Arg Gly Leu Gln Leu Gln Gln Glu Phe Val Asn
Phe305 310 315 320Phe Leu Leu His Tyr Lys Lys Glu Val Ser Val Ser
Asp Gln Met Glu 325 330 335Phe Val Asn Gly Trp Tyr Ile Met Ile Ile
Ile Ser Asp Ile Leu Thr 340 345 350Ile Ile Gly Ser Ile Leu Lys Met
Glu Ile Gln Ala Lys Ser Leu Thr 355 360 365Ser Tyr Asp Val Cys Ser
Ile Leu Leu Gly Thr Ser Thr Met Leu Val 370 375 380Trp Leu Gly Val
Ile Arg Tyr Leu Gly Phe Phe Ala Lys Tyr Asn Leu385 390 395 400Leu
Ile Leu Thr Leu Gln Ala Ala Leu Pro Asn Val Ile Arg Phe Cys 405 410
415Cys Cys Pro Ala Met Ile Tyr Leu Gly Tyr Cys Phe Cys Gly Trp Ile
420 425 430Val Leu Gly Pro Tyr His Asp Lys Phe Arg Ser Leu Asn Met
Val Ser 435 440 445Glu Cys Leu Phe Ser Leu Ile Asn Gly Asp Asp Met
Phe Ala Thr Phe 450 455 460Ala Lys Met Gln Gln Lys Ser Tyr Leu Val
Trp Leu Phe Ser Arg Ile465 470 475 480Tyr Leu Tyr Ser Phe Ile Ser
Leu Phe Ile Tyr Met Ile Leu Ser Leu 485 490 495Phe Ile Ala Leu Ile
Thr Asp Thr Tyr Glu Thr Ile Lys Gln Tyr Gln 500 505 510Gln Asp Gly
Phe Pro Glu Thr Glu Leu Arg Thr Phe Ile Ser Glu Cys 515 520 525Lys
Asp Leu Pro Asn Ser Gly Lys Tyr Arg Leu Glu Asp Asp Pro Pro 530 535
540Val Ser Leu Phe Cys Cys Cys Lys Lys545 55051740DNAHomo sapiens
5atgacagccc cggcgggtcc gcgcggctca gagaccgagc ggcttctgac ccccaacccc
60gggtatggga cccaggcggg gccttcaccg gcccctccga cacccccaga agaggaagac
120cttcgccgtc gtctcaaata ctttttcatg agtccctgcg acaagtttcg
agccaagggc 180cgcaagccct gcaagctgat gctgcaagtg gtcaagatcc
tggtggtcac ggtgcagctc 240atcctgtttg ggctcagtaa tcagctggct
gtgacattcc gggaagagaa caccatcgcc 300ttccgacacc tcttcctgct
gggctactcg gacggagcgg atgacacctt cgcagcctac 360acgcgggagc
agctgtacca ggccatcttc catgctgtgg accagtacct ggcgttgcct
420gacgtgtcac tgggccggta tgcgtatgtc cgtggtgggg gtgacccttg
gaccaatggc 480tcagggcttg ctctctgcca gcggtactac caccgaggcc
acgtggaccc ggccaacgac 540acatttgaca ttgatccgat ggtggttact
gactgcatcc aggtggatcc ccccgagcgg 600ccccctccgc cccccagcga
cgatctcacc ctcttggaaa gcagctccag ttacaagaac 660ctcacgctca
aattccacaa gctggtcaat gtcaccatcc acttccggct gaagaccatt
720aacctccaga gcctcatcaa taatgagatc ccggactgct ataccttcag
cgtcctgatc 780acgtttgaca acaaagcaca cagtgggcgg atccccatca
gcctggagac ccaggcccac 840atccaggagt gtaagcaccc cagtgtcttc
cagcacggag acaacagctt ccggctcctg 900tttgacgtgg tggtcatcct
cacctgctcc ctgtccttcc tcctctgcgc ccgctcactc 960cttcgaggct
tcctgctgca gaacgagttt gtggggttca tgtggcggca gcggggacgg
1020gtcatcagcc tgtgggagcg gctggaattt gtcaatggct ggtacatcct
gctcgtcacc 1080agcgatgtgc tcaccatctc gggcaccatc atgaagatcg
gcatcgaggc caagaacttg 1140gcgagctacg acgtctgcag catcctcctg
ggcacctcga cgctgctggt gtgggtgggc 1200gtgatccgct acctgacctt
cttccacaac tacaatatcc tcatcgccac actgcgggtg 1260gccctgccca
gcgtcatgcg cttctgctgc tgcgtggctg tcatctacct gggctactgc
1320ttctgtggct ggatcgtgct ggggccctat catgtgaagt tccgctcact
ctccatggtg 1380tctgagtgcc tgttctcgct catcaatggg gacgacatgt
ttgtgacgtt cgccgccatg 1440caggcgcagc agggccgcag cagcctggtg
tggctcttct cccagctcta cctttactcc 1500ttcatcagcc tcttcatcta
catggtgctc agcctcttca tcgcgctcat caccggcgcc 1560tacgacacca
tcaagcatcc cggcggcgca ggcgcagagg agagcgagct gcaggcctac
1620atcgcacagt gccaggacag ccccacctcc ggcaagttcc gccgcgggag
cggctcggcc 1680tgcagccttc tctgctgctg cggaagggac ccctcggagg
agcattcgct gctggtgaat 17406580PRTHomo sapiens 6Met Thr Ala Pro Ala
Gly Pro Arg Gly Ser Glu Thr Glu Arg Leu Leu1 5 10 15Thr Pro Asn Pro
Gly Tyr Gly Thr Gln Ala Gly Pro Ser Pro Ala Pro 20 25 30Pro Thr Pro
Pro Glu Glu Glu Asp Leu Arg Arg Arg Leu Lys Tyr Phe 35 40 45Phe Met
Ser Pro Cys Asp Lys Phe Arg Ala Lys Gly Arg Lys Pro Cys 50 55 60Lys
Leu Met Leu Gln Val Val Lys Ile Leu Val Val Thr Val Gln Leu65 70 75
80Ile Leu Phe Gly Leu Ser Asn Gln Leu Ala Val Thr Phe Arg Glu Glu
85 90 95Asn Thr Ile Ala Phe Arg His Leu Phe Leu Leu Gly Tyr Ser Asp
Gly 100 105 110Ala Asp Asp Thr Phe Ala Ala Tyr Thr Arg Glu Gln Leu
Tyr Gln Ala 115 120 125Ile Phe His Ala Val Asp Gln Tyr Leu Ala Leu
Pro Asp Val Ser Leu 130 135 140Gly Arg Tyr Ala Tyr Val Arg Gly Gly
Gly Asp Pro Trp Thr Asn Gly145 150 155 160Ser Gly Leu Ala Leu Cys
Gln Arg Tyr Tyr His Arg Gly His Val Asp 165 170 175Pro Ala Asn Asp
Thr Phe Asp Ile Asp Pro Met Val Val Thr Asp Cys 180 185 190Ile Gln
Val Asp Pro Pro Glu Arg Pro Pro Pro Pro Pro Ser Asp Asp 195 200
205Leu Thr Leu Leu Glu Ser Ser Ser Ser Tyr Lys Asn Leu Thr Leu Lys
210 215 220Phe His Lys Leu Val Asn Val Thr Ile His Phe Arg Leu Lys
Thr Ile225 230 235 240Asn Leu Gln Ser Leu Ile Asn Asn Glu Ile Pro
Asp Cys Tyr Thr Phe 245 250 255Ser Val Leu Ile Thr Phe Asp Asn Lys
Ala His Ser Gly Arg Ile Pro 260 265 270Ile Ser Leu Glu Thr Gln Ala
His Ile Gln Glu Cys Lys His Pro Ser 275 280 285Val Phe Gln His Gly
Asp Asn Ser Phe Arg Leu Leu Phe Asp Val Val 290 295 300Val Ile Leu
Thr Cys Ser Leu Ser Phe Leu Leu Cys Ala Arg Ser Leu305 310 315
320Leu Arg Gly Phe Leu Leu Gln Asn Glu Phe Val Gly Phe Met Trp Arg
325 330 335Gln Arg Gly Arg Val Ile Ser Leu Trp Glu Arg Leu Glu Phe
Val Asn 340 345 350Gly Trp Tyr Ile Leu Leu Val Thr Ser Asp Val Leu
Thr Ile Ser Gly 355 360 365Thr Ile Met Lys Ile Gly Ile Glu Ala Lys
Asn Leu Ala Ser Tyr Asp 370 375 380Val Cys Ser Ile Leu Leu Gly Thr
Ser Thr Leu Leu Val Trp Val Gly385 390 395 400Val Ile Arg Tyr Leu
Thr Phe Phe His Asn Tyr Asn Ile Leu Ile Ala 405 410 415Thr Leu Arg
Val Ala Leu Pro Ser Val Met Arg Phe Cys Cys Cys Val 420 425 430Ala
Val Ile Tyr Leu Gly Tyr Cys Phe Cys Gly Trp Ile Val Leu Gly 435 440
445Pro Tyr His Val Lys Phe Arg Ser Leu Ser Met Val Ser Glu Cys Leu
450 455 460Phe Ser Leu Ile Asn Gly Asp Asp Met Phe Val Thr Phe Ala
Ala Met465 470 475 480Gln Ala Gln Gln Gly Arg Ser Ser Leu Val Trp
Leu Phe Ser Gln Leu 485 490 495Tyr Leu Tyr Ser Phe Ile Ser Leu Phe
Ile Tyr Met Val Leu Ser Leu 500 505 510Phe Ile Ala Leu Ile Thr
Gly Ala Tyr Asp Thr Ile Lys His Pro Gly 515 520 525Gly Ala Gly Ala
Glu Glu Ser Glu Leu Gln Ala Tyr Ile Ala Gln Cys 530 535 540Gln Asp
Ser Pro Thr Ser Gly Lys Phe Arg Arg Gly Ser Gly Ser Ala545 550 555
560Cys Ser Leu Leu Cys Cys Cys Gly Arg Asp Pro Ser Glu Glu His Ser
565 570 575Leu Leu Val Asn 58071698DNAHomo sapiens 7atggcccggc
agccttatcg ttttccccag gcaaggattc cggagagagg atcaggtgtt 60ttcaggttaa
ccgtcagaaa cgcaatggca catcgtgatt ctgagatgaa agaagaatgt
120ctaagggaag acctgaagtt ttacttcatg agcccttgtg aaaaataccg
agccagacgc 180cagattccgt ggaaactggg tttgcagatt ttgaagatag
tcatggtcac cacacagctt 240gttcgttttg gtttaagtaa ccagctggtg
gttgctttca aagaagataa cactgttgct 300tttaagcact tgtttttgaa
aggatattct ggtacagatg aagatgacta cagctgcagt 360gtatatactc
aagaggatgc ctatgagagc atcttttttg ctattaatca gtatcatcag
420ctaaaggaca ttaccctggg gacccttggt tatggagaaa atgaagacaa
tagaattggc 480ttaaaagtct gtaagcagca ttacaagaaa gggaccatgt
ttccttctaa tgagacactg 540aatattgaca acgacgttga gctcgattgt
gttcaattag accttcagga cctctccaag 600aagcctccgg actggaagaa
ctcatcattc ttcagactgg aattttatcg gctcttacag 660gttgaaatct
cctttcatct taaaggcatt gacctacaga caattcattc ccgtgagtta
720ccagactgtt atgtctttca gaatacgatt atctttgaca ataaagctca
cagtggcaaa 780atcaaaatct attttgacag tgatgccaaa attgaagaat
gtaaagactt gaacatattt 840ggatctactc agaaaaatgc tcagtatgtc
ctggtgtttg atgcatttgt cattgtgatt 900tgcttggcat ctcttattct
gtgtacaaga tccattgttc ttgctctaag gttacggaag 960agatttctaa
atttcttcct ggagaagtac aagcggcctg tgtgtgacac cgaccagtgg
1020gagttcatca acggctggta tgtcctggtg attatcagcg acctaatgac
aatcattggc 1080tccatattaa aaatggaaat caaagcaaag aatctcacaa
actatgatct ctgcagcatt 1140tttcttggaa cctctacgct cttggtttgg
gttggagtca tcagatacct gggttatttc 1200caggcatata atgtgctgat
tttaacaatg caggcctcac tgccaaaagt tcttcggttt 1260tgtgcttgtg
ctggtatgat ttatctgggt tacacattct gtggctggat tgtcttagga
1320ccataccatg acaagtttga aaatctgaac acagttgctg agtgtctgtt
ttctctggtc 1380aacggtgatg acatgtttgc aacctttgcc caaatccagc
agaagagcat cttggtgtgg 1440ctgttcagtc gtctgtattt atattccttc
atcagccttt ttatatatat gattctcagt 1500ctttttattg cacttattac
agattcttat gacaccatta agaaattcca acagaatggg 1560tttcctgaaa
cggatttgca ggaattcctg aaggaatgca gtagcaaaga agagtatcag
1620aaagagtcct cagccttcct gtcctgcatc tgctgtcgga ggaggaaaag
aagtgatgat 1680cacttgatac ctattagc 16988566PRTHomo sapiens 8Met Ala
Arg Gln Pro Tyr Arg Phe Pro Gln Ala Arg Ile Pro Glu Arg1 5 10 15Gly
Ser Gly Val Phe Arg Leu Thr Val Arg Asn Ala Met Ala His Arg 20 25
30Asp Ser Glu Met Lys Glu Glu Cys Leu Arg Glu Asp Leu Lys Phe Tyr
35 40 45Phe Met Ser Pro Cys Glu Lys Tyr Arg Ala Arg Arg Gln Ile Pro
Trp 50 55 60Lys Leu Gly Leu Gln Ile Leu Lys Ile Val Met Val Thr Thr
Gln Leu65 70 75 80Val Arg Phe Gly Leu Ser Asn Gln Leu Val Val Ala
Phe Lys Glu Asp 85 90 95Asn Thr Val Ala Phe Lys His Leu Phe Leu Lys
Gly Tyr Ser Gly Thr 100 105 110Asp Glu Asp Asp Tyr Ser Cys Ser Val
Tyr Thr Gln Glu Asp Ala Tyr 115 120 125Glu Ser Ile Phe Phe Ala Ile
Asn Gln Tyr His Gln Leu Lys Asp Ile 130 135 140Thr Leu Gly Thr Leu
Gly Tyr Gly Glu Asn Glu Asp Asn Arg Ile Gly145 150 155 160Leu Lys
Val Cys Lys Gln His Tyr Lys Lys Gly Thr Met Phe Pro Ser 165 170
175Asn Glu Thr Leu Asn Ile Asp Asn Asp Val Glu Leu Asp Cys Val Gln
180 185 190Leu Asp Leu Gln Asp Leu Ser Lys Lys Pro Pro Asp Trp Lys
Asn Ser 195 200 205Ser Phe Phe Arg Leu Glu Phe Tyr Arg Leu Leu Gln
Val Glu Ile Ser 210 215 220Phe His Leu Lys Gly Ile Asp Leu Gln Thr
Ile His Ser Arg Glu Leu225 230 235 240Pro Asp Cys Tyr Val Phe Gln
Asn Thr Ile Ile Phe Asp Asn Lys Ala 245 250 255His Ser Gly Lys Ile
Lys Ile Tyr Phe Asp Ser Asp Ala Lys Ile Glu 260 265 270Glu Cys Lys
Asp Leu Asn Ile Phe Gly Ser Thr Gln Lys Asn Ala Gln 275 280 285Tyr
Val Leu Val Phe Asp Ala Phe Val Ile Val Ile Cys Leu Ala Ser 290 295
300Leu Ile Leu Cys Thr Arg Ser Ile Val Leu Ala Leu Arg Leu Arg
Lys305 310 315 320Arg Phe Leu Asn Phe Phe Leu Glu Lys Tyr Lys Arg
Pro Val Cys Asp 325 330 335Thr Asp Gln Trp Glu Phe Ile Asn Gly Trp
Tyr Val Leu Val Ile Ile 340 345 350Ser Asp Leu Met Thr Ile Ile Gly
Ser Ile Leu Lys Met Glu Ile Lys 355 360 365Ala Lys Asn Leu Thr Asn
Tyr Asp Leu Cys Ser Ile Phe Leu Gly Thr 370 375 380Ser Thr Leu Leu
Val Trp Val Gly Val Ile Arg Tyr Leu Gly Tyr Phe385 390 395 400Gln
Ala Tyr Asn Val Leu Ile Leu Thr Met Gln Ala Ser Leu Pro Lys 405 410
415Val Leu Arg Phe Cys Ala Cys Ala Gly Met Ile Tyr Leu Gly Tyr Thr
420 425 430Phe Cys Gly Trp Ile Val Leu Gly Pro Tyr His Asp Lys Phe
Glu Asn 435 440 445Leu Asn Thr Val Ala Glu Cys Leu Phe Ser Leu Val
Asn Gly Asp Asp 450 455 460Met Phe Ala Thr Phe Ala Gln Ile Gln Gln
Lys Ser Ile Leu Val Trp465 470 475 480Leu Phe Ser Arg Leu Tyr Leu
Tyr Ser Phe Ile Ser Leu Phe Ile Tyr 485 490 495Met Ile Leu Ser Leu
Phe Ile Ala Leu Ile Thr Asp Ser Tyr Asp Thr 500 505 510Ile Lys Lys
Phe Gln Gln Asn Gly Phe Pro Glu Thr Asp Leu Gln Glu 515 520 525Phe
Leu Lys Glu Cys Ser Ser Lys Glu Glu Tyr Gln Lys Glu Ser Ser 530 535
540Ala Phe Leu Ser Cys Ile Cys Cys Arg Arg Arg Lys Arg Ser Asp
Asp545 550 555 560His Leu Ile Pro Ile Ser 56591659DNAMus musculus
9atggcaaatc ccgaggtgct ggttagcagc tgcagagctc gccaagatga aagcccctgc
60actttccacc cgagctcgtc cccgtcagag cagcttctct tagaagacca gatgaggcgg
120aaactcaagt tcttttttat gaatccttgt gagaagttct gggctcgggg
taggaagcca 180tggaaacttg ccatacagat tctgaaaatc gcgatggtga
ctatccagct ggttctgttt 240ggactaagta accagatggt agtagctttc
aaagaggaga acactatagc cttcaaacac 300ctcttcctaa agggctacat
ggatcgaatg gacgacacct atgcagtgta cactcagagt 360gaagtgtatg
accagatcat ctttgcagtg acccagtact tgcagcttca gaacatctcc
420gtgggcaatc acgcttatga gaacaagggg actaagcagt cggcgatggc
aatctgtcag 480cacttctaca ggcaaggaac catctgcccc gggaacgaca
cctttgacat cgatccagaa 540gttgaaacag aatgtttcct tgtagagcca
gatgaagctt cccaccttgg aacgcctgga 600gaaaataaac tcaacctgag
cctggacttc cacagacttc tgacggtgga gctccagttt 660aagctcaaag
ccatcaatct gcagacagtt cgacaccagg agcttcctga ctgttacgac
720tttacgctga ctataacatt cgacaacaag gctcacagtg gaagaatcaa
aataagctta 780gacaacgaca tttctatcaa agaatgcaaa gactggcatg
tgtctggatc aattcagaag 840aacacacact acatgatgat ctttgatgcc
tttgtcattc tgacctgctt ggcctcactg 900gtgctgtgtg ccaggtctgt
gattaggggt cttcagcttc agcaggagtt tgtcaacttc 960ttccttcttc
actacaagaa ggaagtttcg gcctctgatc agatggagtt catcaacggg
1020tggtacatta tgatcatcat tagtgacata ttgacaatcg ttggatcagt
tctgaaaatg 1080gaaatccaag ccaagagtct cacaagctat gatgtctgca
gcatacttct cgggacgtca 1140actatgctcg tgtggcttgg agttatccga
tacctgggtt tctttgcgaa gtacaatctc 1200cttattctga ccctccaggc
agcgctgccc aacgtcatga ggttctgttg ctgcgctgct 1260atgatctatc
taggctattg cttttgcgga tggattgtgc tgggccctta ccatgagaag
1320ttccgttccc tgaacagggt ctccgagtgc ctgttctcgc tgataaacgg
agacgatatg 1380ttttccacat ttgcgaaaat gcagcagaag agttacctgg
tgtggctgtt cagccgagtc 1440tacctgtact cgttcatcag cctcttcatt
tacatgattc tgagcctttt catcgcgctc 1500atcacagaca catacgaaac
aattaagcac taccagcaag atggcttccc agagacggaa 1560cttcgaaagt
ttatagcgga atgcaaagac ctccccaact ccggaaaata cagattagaa
1620gatgaccctc cgggttcttt actctgctgc tgcaaaaag 165910553PRTMus
musculus 10Met Ala Asn Pro Glu Val Leu Val Ser Ser Cys Arg Ala Arg
Gln Asp1 5 10 15Glu Ser Pro Cys Thr Phe His Pro Ser Ser Ser Pro Ser
Glu Gln Leu 20 25 30Leu Leu Glu Asp Gln Met Arg Arg Lys Leu Lys Phe
Phe Phe Met Asn 35 40 45Pro Cys Glu Lys Phe Trp Ala Arg Gly Arg Lys
Pro Trp Lys Leu Ala 50 55 60Ile Gln Ile Leu Lys Ile Ala Met Val Thr
Ile Gln Leu Val Leu Phe65 70 75 80Gly Leu Ser Asn Gln Met Val Val
Ala Phe Lys Glu Glu Asn Thr Ile 85 90 95Ala Phe Lys His Leu Phe Leu
Lys Gly Tyr Met Asp Arg Met Asp Asp 100 105 110Thr Tyr Ala Val Tyr
Thr Gln Ser Glu Val Tyr Asp Gln Ile Ile Phe 115 120 125Ala Val Thr
Gln Tyr Leu Gln Leu Gln Asn Ile Ser Val Gly Asn His 130 135 140Ala
Tyr Glu Asn Lys Gly Thr Lys Gln Ser Ala Met Ala Ile Cys Gln145 150
155 160His Phe Tyr Arg Gln Gly Thr Ile Cys Pro Gly Asn Asp Thr Phe
Asp 165 170 175Ile Asp Pro Glu Val Glu Thr Glu Cys Phe Leu Val Glu
Pro Asp Glu 180 185 190Ala Ser His Leu Gly Thr Pro Gly Glu Asn Lys
Leu Asn Leu Ser Leu 195 200 205Asp Phe His Arg Leu Leu Thr Val Glu
Leu Gln Phe Lys Leu Lys Ala 210 215 220Ile Asn Leu Gln Thr Val Arg
His Gln Glu Leu Pro Asp Cys Tyr Asp225 230 235 240Phe Thr Leu Thr
Ile Thr Phe Asp Asn Lys Ala His Ser Gly Arg Ile 245 250 255Lys Ile
Ser Leu Asp Asn Asp Ile Ser Ile Lys Glu Cys Lys Asp Trp 260 265
270His Val Ser Gly Ser Ile Gln Lys Asn Thr His Tyr Met Met Ile Phe
275 280 285Asp Ala Phe Val Ile Leu Thr Cys Leu Ala Ser Leu Val Leu
Cys Ala 290 295 300Arg Ser Val Ile Arg Gly Leu Gln Leu Gln Gln Glu
Phe Val Asn Phe305 310 315 320Phe Leu Leu His Tyr Lys Lys Glu Val
Ser Ala Ser Asp Gln Met Glu 325 330 335Phe Ile Asn Gly Trp Tyr Ile
Met Ile Ile Ile Ser Asp Ile Leu Thr 340 345 350Ile Val Gly Ser Val
Leu Lys Met Glu Ile Gln Ala Lys Ser Leu Thr 355 360 365Ser Tyr Asp
Val Cys Ser Ile Leu Leu Gly Thr Ser Thr Met Leu Val 370 375 380Trp
Leu Gly Val Ile Arg Tyr Leu Gly Phe Phe Ala Lys Tyr Asn Leu385 390
395 400Leu Ile Leu Thr Leu Gln Ala Ala Leu Pro Asn Val Met Arg Phe
Cys 405 410 415Cys Cys Ala Ala Met Ile Tyr Leu Gly Tyr Cys Phe Cys
Gly Trp Ile 420 425 430Val Leu Gly Pro Tyr His Glu Lys Phe Arg Ser
Leu Asn Arg Val Ser 435 440 445Glu Cys Leu Phe Ser Leu Ile Asn Gly
Asp Asp Met Phe Ser Thr Phe 450 455 460Ala Lys Met Gln Gln Lys Ser
Tyr Leu Val Trp Leu Phe Ser Arg Val465 470 475 480Tyr Leu Tyr Ser
Phe Ile Ser Leu Phe Ile Tyr Met Ile Leu Ser Leu 485 490 495Phe Ile
Ala Leu Ile Thr Asp Thr Tyr Glu Thr Ile Lys His Tyr Gln 500 505
510Gln Asp Gly Phe Pro Glu Thr Glu Leu Arg Lys Phe Ile Ala Glu Cys
515 520 525Lys Asp Leu Pro Asn Ser Gly Lys Tyr Arg Leu Glu Asp Asp
Pro Pro 530 535 540Gly Ser Leu Leu Cys Cys Cys Lys Lys545
550111773DNAGallus gallus 11atgatcaccc gtggtttccg ttcaggcttt
tacggctgta gcaatcgata tagtggtgct 60aagtgccagt tagttgctca gacgtttgtc
atttcagcaa tggagactcc tgaagtggct 120gtaagcagct gcagtgctcg
ggatgacgaa gggctctgca gctacggaca acacctattg 180ctgccacaag
agctggtggc ggaagaccag ctgaggagga agctgaagtt cttcttcatg
240aacccatgtg aaaaattctg ggctcggggc agaaaacctt ggaaacttgg
gattcagctg 300ctcaaaatag caatggttac cattcagctg gtgctttttg
gattgagcaa tcaaatggtg 360gttgctttca aagaagagaa cactattgca
ttcaaacatc tcttcttgaa agggtacatg 420gacagaatgg atgataccta
tgcggtatac acacagacag atgtctatga ccaaatattc 480tttgccatca
atcagtactt acagttgccc aacatttctg ttggaaacca tgcttatgag
540aagaaaggag cagaagagac agctctggct gtatgtcaac agttctacaa
gcaaggaacc 600atctgtcctg gaaatgacac ctttgatata gacccagaga
ttgtgactga ctgcttgtac 660attgagccga tgatgtcttt agacaacaga
acagtgggaa agcacaattt gaatttcact 720ctggatttcc acaggctcgt
ggcagtgcaa ctcatgttca atctgaaggc aatcaacctc 780cagaccgtcc
gtcaccacga gctccctgac tgttacgatt tcaccctgac gatagtgttt
840gataataaag cccacagtgg aagaatcaaa atcagtctag acaacgacat
agagatcagg 900gaatgtaaag actggcacgt ttctggatca atacagaaga
atacgcatta catgatgatc 960ttcgatgctt ttgtcatact gatctgtctg
agctcattga tcctttgcac tcgatcagta 1020gtcaaaggaa ttcggctcca
aagagaattt gtaagttttt tcctatatta ttacaagaaa 1080gaggtatctt
acaatgatca gatggaattt gtcaatggct ggtatatcct cattatggtt
1140agtgatgtcc tcactatcgt tggatcaact ctcaaaatgg agatacaggc
caagagtctg 1200acaagttacg acgtctgtag catactctta ggaacatcca
ctatgctggt gtggcttgga 1260gtcattcgct acctcggttt ctttcagaag
tataatcttc tcattctaac gctgcgagca 1320gcactaccca acgtcatgag
gttctgctgt tgtgctgcta tgatctatct aggttattgt 1380ttctgcggat
ggattgtact ggggccatac cacgtgaagt tccgttctct gaatgtggtt
1440tctgaatgcc tcttttcatt gataaatgga gatgacatgt ttgccacttt
tgcaaaaatg 1500cagcagaaaa gttacttggt ttggttattc agtagaatct
acctctactc cttcatcagc 1560ctgttcatct acatggtgct aagtctcttc
attgcactca ttacagatac atatgaaact 1620atcaagcact accaacaaga
tggctttcca gagacagaac ttcagagatt tatatcacag 1680tgcaaagact
taccaaactc tggaaggtac agattagaag aggaaggttc tgtatctctc
1740ttctgttgtt gcagtggtcc tagtgaacat atc 177312591PRTGallus gallus
12Met Ile Thr Arg Gly Phe Arg Ser Gly Phe Tyr Gly Cys Ser Asn Arg1
5 10 15Tyr Ser Gly Ala Lys Cys Gln Leu Val Ala Gln Thr Phe Val Ile
Ser 20 25 30Ala Met Glu Thr Pro Glu Val Ala Val Ser Ser Cys Ser Ala
Arg Asp 35 40 45Asp Glu Gly Leu Cys Ser Tyr Gly Gln His Leu Leu Leu
Pro Gln Glu 50 55 60Leu Val Ala Glu Asp Gln Leu Arg Arg Lys Leu Lys
Phe Phe Phe Met65 70 75 80Asn Pro Cys Glu Lys Phe Trp Ala Arg Gly
Arg Lys Pro Trp Lys Leu 85 90 95Gly Ile Gln Leu Leu Lys Ile Ala Met
Val Thr Ile Gln Leu Val Leu 100 105 110Phe Gly Leu Ser Asn Gln Met
Val Val Ala Phe Lys Glu Glu Asn Thr 115 120 125Ile Ala Phe Lys His
Leu Phe Leu Lys Gly Tyr Met Asp Arg Met Asp 130 135 140Asp Thr Tyr
Ala Val Tyr Thr Gln Thr Asp Val Tyr Asp Gln Ile Phe145 150 155
160Phe Ala Ile Asn Gln Tyr Leu Gln Leu Pro Asn Ile Ser Val Gly Asn
165 170 175His Ala Tyr Glu Lys Lys Gly Ala Glu Glu Thr Ala Leu Ala
Val Cys 180 185 190Gln Gln Phe Tyr Lys Gln Gly Thr Ile Cys Pro Gly
Asn Asp Thr Phe 195 200 205Asp Ile Asp Pro Glu Ile Val Thr Asp Cys
Leu Tyr Ile Glu Pro Met 210 215 220Met Ser Leu Asp Asn Arg Thr Val
Gly Lys His Asn Leu Asn Phe Thr225 230 235 240Leu Asp Phe His Arg
Leu Val Ala Val Gln Leu Met Phe Asn Leu Lys 245 250 255Ala Ile Asn
Leu Gln Thr Val Arg His His Glu Leu Pro Asp Cys Tyr 260 265 270Asp
Phe Thr Leu Thr Ile Val Phe Asp Asn Lys Ala His Ser Gly Arg 275 280
285Ile Lys Ile Ser Leu Asp Asn Asp Ile Glu Ile Arg Glu Cys Lys Asp
290 295 300Trp His Val Ser Gly Ser Ile Gln Lys Asn Thr His Tyr Met
Met Ile305 310 315 320Phe Asp Ala Phe Val Ile Leu Ile Cys Leu Ser
Ser Leu Ile Leu Cys 325 330 335Thr Arg Ser Val Val Lys Gly Ile Arg
Leu Gln Arg Glu Phe Val Ser 340 345 350Phe Phe Leu Tyr Tyr Tyr Lys
Lys Glu Val Ser Tyr Asn Asp Gln Met 355 360 365Glu Phe Val Asn Gly
Trp Tyr Ile Leu Ile Met Val Ser Asp Val Leu 370 375 380Thr Ile Val
Gly Ser Thr Leu Lys Met Glu Ile Gln Ala Lys Ser Leu385 390 395
400Thr Ser Tyr Asp Val Cys Ser Ile Leu Leu Gly Thr Ser Thr Met Leu
405 410
415Val Trp Leu Gly Val Ile Arg Tyr Leu Gly Phe Phe Gln Lys Tyr Asn
420 425 430Leu Leu Ile Leu Thr Leu Arg Ala Ala Leu Pro Asn Val Met
Arg Phe 435 440 445Cys Cys Cys Ala Ala Met Ile Tyr Leu Gly Tyr Cys
Phe Cys Gly Trp 450 455 460Ile Val Leu Gly Pro Tyr His Val Lys Phe
Arg Ser Leu Asn Val Val465 470 475 480Ser Glu Cys Leu Phe Ser Leu
Ile Asn Gly Asp Asp Met Phe Ala Thr 485 490 495Phe Ala Lys Met Gln
Gln Lys Ser Tyr Leu Val Trp Leu Phe Ser Arg 500 505 510Ile Tyr Leu
Tyr Ser Phe Ile Ser Leu Phe Ile Tyr Met Val Leu Ser 515 520 525Leu
Phe Ile Ala Leu Ile Thr Asp Thr Tyr Glu Thr Ile Lys His Tyr 530 535
540Gln Gln Asp Gly Phe Pro Glu Thr Glu Leu Gln Arg Phe Ile Ser
Gln545 550 555 560Cys Lys Asp Leu Pro Asn Ser Gly Arg Tyr Arg Leu
Glu Glu Glu Gly 565 570 575Ser Val Ser Leu Phe Cys Cys Cys Ser Gly
Pro Ser Glu His Ile 580 585 590132424DNACanis familiaris
13atgacccctt ttggcagctt ggcttctgca aaggcttcaa actcaagagc tgcctggaag
60attgtggtgg attcattcag ctcgcagcac acgccccagg tgggcactgg tggtccccag
120gaatcagaca aggcccttac cttcggagag ttaacgttct cccactcatc
tccattcttc 180atctgcgcca gcccttctct cccaccgttg cacagcagtg
ggctagacga tgagccatac 240tgctggacag gttttcactg catcaagtac
ctcgcgggcc cagcgagtgt cccaaactcc 300cttgaaagag ggagtaagat
attggtttcc caagcctcct ttcccatccg gacctcccct 360tacctgacac
tgctaagccg aggggaaaag aagcctctct gcagttccgt ggagaaaagg
420cctttggggg ttttggagat gggaagtctg actctcctct cggaagagct
caaacggcag 480ctccccggca cgctgttgct aggacaacct ccgttgctaa
gggaaagagg cggctcctct 540gctgagattg acaagacgcc gctaccaata
gggcgtctac tctccggccg cctcttcaag 600gccgcacttg tgattggctg
ctgtcgtgct gacgtcacgc aactcgaacc gccgagagat 660cctgggtttt
cgcccgctcg gcaggaggtg tgcggtttgg gttcccgcgt ggagggtgct
720gctggctcga atgtaaacaa tctttttttt tttctccccc tagagatggc
aaatcctacg 780gttgttataa gtagctgcag ctctcatgaa gaggaaaatc
gttgcacttt tagccagcac 840acatcgccct ctgaggagct tctgttagaa
gaccagataa ggcgaaaact caaatttttt 900ttcatgaatc cttgtgaaaa
gttctgggct cgaggtagaa aaccatggaa gcttgccata 960caaattctaa
aaattgcaat ggtgactatc cagctggtct tttttgggct aagtaaccag
1020atggttgtag ctttcaagga agaaaacact atagcattca aacacctctt
cttaaaagga 1080tatatggacc gaatggatga cacatatgca gtgtacacac
aacgtgatgt atatgatcag 1140atcatctttg cagtgaacca gtacttgctt
ctacgcaata cctcggttgg gaatcatgct 1200tatgagaaca aggggacgga
acagtctgct atggcaatct gtcagcactt ctacaagcag 1260ggaaacatct
gtcctggaaa tgataccttc gacattgatc cagagattga aactgagtgt
1320ttctctgtag agccagctga gcctttccac gtcggaacac tggaagaaaa
taaactcaac 1380ttaacgctgg actttcacag actcctcacg gtggacctgc
agtttaagct gaaggccatt 1440aatctgcaga ccattcggca tcacgagctc
cctgactgtt atgactttac tctcactata 1500acatttgaca ataaggccca
tagtggaaga attaagataa gtttagataa tgatatttcc 1560atcagagaat
gtaaagactg gcatgtatcg ggatcaattc agaagaacac tcactacatg
1620atgatctttg atgcctttgt tattctgaca tgcttggctt cactaaccct
gtgccttcga 1680tctgtaatta gaggacttca gcttcaacag gaatttgtca
attttttcct cctccattat 1740aagaaggaag tttctgtttc tgatcgaatg
gaatttgtca atggatggta cattatgatt 1800attattagtg acatgttgac
aattattgga tcaattctga aaatggaaat tcaagctaag 1860agtctaacaa
gttatgatgt ttgtagcata cttcttggga cttccaccat gcttgtgtgg
1920cttggagtta ttcgatacct cggcttcttt cagaagtaca atctccttat
tctgaccctg 1980caggcagcac tgcccagtgt catcaggttc tgttgctgtg
ccgctatgat ttatttaggc 2040tattgcttct gtggatggat tgtgctgggg
ccgtaccatg ataagttccg ttctctgaac 2100atggtctctg agtgcctttt
ctctctgata aatggagatg atatgtttgc cacatttgca 2160aaaatgcaac
aaaaaagtta cttggtctgg ctgtttagca gaatttatct ctactcattc
2220atcagcctct ttatatatat gattttaagt cttttcatcg cactgatcac
tgatacgtat 2280gaaacaatta agcattacca acaagatggc tttccagaga
ctgaacttcg tacatttata 2340tcagagtgca aagatctacc caattctgga
aaatacagat tagaagatga cactccaata 2400tctatattct gctgttgtaa aaag
242414808PRTCanis familiaris 14Met Thr Pro Phe Gly Ser Leu Ala Ser
Ala Lys Ala Ser Asn Ser Arg1 5 10 15Ala Ala Trp Lys Ile Val Val Asp
Ser Phe Ser Ser Gln His Thr Pro 20 25 30Gln Val Gly Thr Gly Gly Pro
Gln Glu Ser Asp Lys Ala Leu Thr Phe 35 40 45Gly Glu Leu Thr Phe Ser
His Ser Ser Pro Phe Phe Ile Cys Ala Ser 50 55 60Pro Ser Leu Pro Pro
Leu His Ser Ser Gly Leu Asp Asp Glu Pro Tyr65 70 75 80Cys Trp Thr
Gly Phe His Cys Ile Lys Tyr Leu Ala Gly Pro Ala Ser 85 90 95Val Pro
Asn Ser Leu Glu Arg Gly Ser Lys Ile Leu Val Ser Gln Ala 100 105
110Ser Phe Pro Ile Arg Thr Ser Pro Tyr Leu Thr Leu Leu Ser Arg Gly
115 120 125Glu Lys Lys Pro Leu Cys Ser Ser Val Glu Lys Arg Pro Leu
Gly Val 130 135 140Leu Glu Met Gly Ser Leu Thr Leu Leu Ser Glu Glu
Leu Lys Arg Gln145 150 155 160Leu Pro Gly Thr Leu Leu Leu Gly Gln
Pro Pro Leu Leu Arg Glu Arg 165 170 175Gly Gly Ser Ser Ala Glu Ile
Asp Lys Thr Pro Leu Pro Ile Gly Arg 180 185 190Leu Leu Ser Gly Arg
Leu Phe Lys Ala Ala Leu Val Ile Gly Cys Cys 195 200 205Arg Ala Asp
Val Thr Gln Leu Glu Pro Pro Arg Asp Pro Gly Phe Ser 210 215 220Pro
Ala Arg Gln Glu Val Cys Gly Leu Gly Ser Arg Val Glu Gly Ala225 230
235 240Ala Gly Ser Asn Val Asn Asn Leu Phe Phe Phe Leu Pro Leu Glu
Met 245 250 255Ala Asn Pro Thr Val Val Ile Ser Ser Cys Ser Ser His
Glu Glu Glu 260 265 270Asn Arg Cys Thr Phe Ser Gln His Thr Ser Pro
Ser Glu Glu Leu Leu 275 280 285Leu Glu Asp Gln Ile Arg Arg Lys Leu
Lys Phe Phe Phe Met Asn Pro 290 295 300Cys Glu Lys Phe Trp Ala Arg
Gly Arg Lys Pro Trp Lys Leu Ala Ile305 310 315 320Gln Ile Leu Lys
Ile Ala Met Val Thr Ile Gln Leu Val Phe Phe Gly 325 330 335Leu Ser
Asn Gln Met Val Val Ala Phe Lys Glu Glu Asn Thr Ile Ala 340 345
350Phe Lys His Leu Phe Leu Lys Gly Tyr Met Asp Arg Met Asp Asp Thr
355 360 365Tyr Ala Val Tyr Thr Gln Arg Asp Val Tyr Asp Gln Ile Ile
Phe Ala 370 375 380Val Asn Gln Tyr Leu Leu Leu Arg Asn Thr Ser Val
Gly Asn His Ala385 390 395 400Tyr Glu Asn Lys Gly Thr Glu Gln Ser
Ala Met Ala Ile Cys Gln His 405 410 415Phe Tyr Lys Gln Gly Asn Ile
Cys Pro Gly Asn Asp Thr Phe Asp Ile 420 425 430Asp Pro Glu Ile Glu
Thr Glu Cys Phe Ser Val Glu Pro Ala Glu Pro 435 440 445Phe His Val
Gly Thr Leu Glu Glu Asn Lys Leu Asn Leu Thr Leu Asp 450 455 460Phe
His Arg Leu Leu Thr Val Asp Leu Gln Phe Lys Leu Lys Ala Ile465 470
475 480Asn Leu Gln Thr Ile Arg His His Glu Leu Pro Asp Cys Tyr Asp
Phe 485 490 495Thr Leu Thr Ile Thr Phe Asp Asn Lys Ala His Ser Gly
Arg Ile Lys 500 505 510Ile Ser Leu Asp Asn Asp Ile Ser Ile Arg Glu
Cys Lys Asp Trp His 515 520 525Val Ser Gly Ser Ile Gln Lys Asn Thr
His Tyr Met Met Ile Phe Asp 530 535 540Ala Phe Val Ile Leu Thr Cys
Leu Ala Ser Leu Thr Leu Cys Leu Arg545 550 555 560Ser Val Ile Arg
Gly Leu Gln Leu Gln Gln Glu Phe Val Asn Phe Phe 565 570 575Leu Leu
His Tyr Lys Lys Glu Val Ser Val Ser Asp Arg Met Glu Phe 580 585
590Val Asn Gly Trp Tyr Ile Met Ile Ile Ile Ser Asp Met Leu Thr Ile
595 600 605Ile Gly Ser Ile Leu Lys Met Glu Ile Gln Ala Lys Ser Leu
Thr Ser 610 615 620Tyr Asp Val Cys Ser Ile Leu Leu Gly Thr Ser Thr
Met Leu Val Trp625 630 635 640Leu Gly Val Ile Arg Tyr Leu Gly Phe
Phe Gln Lys Tyr Asn Leu Leu 645 650 655Ile Leu Thr Leu Gln Ala Ala
Leu Pro Ser Val Ile Arg Phe Cys Cys 660 665 670Cys Ala Ala Met Ile
Tyr Leu Gly Tyr Cys Phe Cys Gly Trp Ile Val 675 680 685Leu Gly Pro
Tyr His Asp Lys Phe Arg Ser Leu Asn Met Val Ser Glu 690 695 700Cys
Leu Phe Ser Leu Ile Asn Gly Asp Asp Met Phe Ala Thr Phe Ala705 710
715 720Lys Met Gln Gln Lys Ser Tyr Leu Val Trp Leu Phe Ser Arg Ile
Tyr 725 730 735Leu Tyr Ser Phe Ile Ser Leu Phe Ile Tyr Met Ile Leu
Ser Leu Phe 740 745 750Ile Ala Leu Ile Thr Asp Thr Tyr Glu Thr Ile
Lys His Tyr Gln Gln 755 760 765Asp Gly Phe Pro Glu Thr Glu Leu Arg
Thr Phe Ile Ser Glu Cys Lys 770 775 780Asp Leu Pro Asn Ser Gly Lys
Tyr Arg Leu Glu Asp Asp Thr Pro Ile785 790 795 800Ser Ile Phe Cys
Cys Cys Lys Lys 805151518DNADanio rerio 15atgtctgacc gagcgtcaca
cactcatgaa agcgcaacac ttctggaccc ggagtgtgtg 60gaaagcttaa ggagaaaact
caagtatttc ttcatgagtc cgtgtcagaa atacagcact 120agaggacgga
taccatggaa gatgatgctt cagatactca agatttgttt agtattcatc
180tacctggtct cttttggatt gagcaacgag atgatggtga cgttcaaaga
ggaaaatctc 240atcgccttca agcacttctt tctgaaaaac tacaaggaca
gcaataaaca ttacgccttg 300tacacaaaac atgaagttca cgaccacatc
ctctacacca tcagacggta tctacagcta 360caaaacctga cgattggcaa
tcaagcgctg gagatgatcg atggtctggc gactcctctg 420tctctctgtc
agcagttgta tcgacatgcg cgcgtcgtgc cgtctaatga gacgtttgaa
480atcgatccac atgtagagac agagtgtgtt tctgtgtatc ccctttctcc
catcacgact 540gacagtctgg aaaactccct gaacttgact ttagattttc
aaaggttgtt agcggtaaac 600atttatctga agatcaaggc tatcaacatt
cagacggttc gccatcaaga gttaccagac 660tgctacgact tcagcattaa
tatcatgttt gacaatcgtg cacacagcgg acagatcaag 720atctctctca
gcagcggcgt gcagataaac gtctgtaagg actggaacat ttctggctca
780agtaagttga acagccactt tgcgctgatt gtggtgtttg actgtttgat
catctgcttc 840tgtctgctgt cactcatcct ctgcacgcgc tcagtccaca
caggatttct cctacagact 900gaatacagaa gattcatgtc cagtcagcac
agtaaaagcg tctcatggtc tgagaggctg 960gagttcatca acggctggta
catcctcatc atcatcagcg atgcgctgac tattgcaggc 1020tcaatcctca
aaatctgcat acagagcaaa gaactgacga gctatgacgt gtgcagtatt
1080ctgctgggca ctgcaacaat gctggtgtgg attggagtaa tgcgctacct
cagtttcttc 1140cagaaatatt atatcctcat cctcaccctg aaggctgcac
ttcccaatgt gattcgattc 1200tccatctgcg ctgttatgat ctacctgagt
tactgcttct gcggatggat cgttttgggg 1260ccacaccatg aaaattttcg
cacattcagt agggttgctg gctgtctttt ctccatgatt 1320aatggggatg
aaatctactc cacgttcacc aagctccggg aatacagcac tctggtgtgg
1380ctgttcagca gactctacgt ctacagcttc atcccggtct tcacatacat
ggttctgagt 1440gtcttcatcg ccctcatcac agacacgtat gaaaccatca
gggtgagtta tttcagcttc 1500agtgagagta gctgcaaa 151816506PRTDanio
rerio 16Met Ser Asp Arg Ala Ser His Thr His Glu Ser Ala Thr Leu Leu
Asp1 5 10 15Pro Glu Cys Val Glu Ser Leu Arg Arg Lys Leu Lys Tyr Phe
Phe Met 20 25 30Ser Pro Cys Gln Lys Tyr Ser Thr Arg Gly Arg Ile Pro
Trp Lys Met 35 40 45Met Leu Gln Ile Leu Lys Ile Cys Leu Val Phe Ile
Tyr Leu Val Ser 50 55 60Phe Gly Leu Ser Asn Glu Met Met Val Thr Phe
Lys Glu Glu Asn Leu65 70 75 80Ile Ala Phe Lys His Phe Phe Leu Lys
Asn Tyr Lys Asp Ser Asn Lys 85 90 95His Tyr Ala Leu Tyr Thr Lys His
Glu Val His Asp His Ile Leu Tyr 100 105 110Thr Ile Arg Arg Tyr Leu
Gln Leu Gln Asn Leu Thr Ile Gly Asn Gln 115 120 125Ala Leu Glu Met
Ile Asp Gly Leu Ala Thr Pro Leu Ser Leu Cys Gln 130 135 140Gln Leu
Tyr Arg His Ala Arg Val Val Pro Ser Asn Glu Thr Phe Glu145 150 155
160Ile Asp Pro His Val Glu Thr Glu Cys Val Ser Val Tyr Pro Leu Ser
165 170 175Pro Ile Thr Thr Asp Ser Leu Glu Asn Ser Leu Asn Leu Thr
Leu Asp 180 185 190Phe Gln Arg Leu Leu Ala Val Asn Ile Tyr Leu Lys
Ile Lys Ala Ile 195 200 205Asn Ile Gln Thr Val Arg His Gln Glu Leu
Pro Asp Cys Tyr Asp Phe 210 215 220Ser Ile Asn Ile Met Phe Asp Asn
Arg Ala His Ser Gly Gln Ile Lys225 230 235 240Ile Ser Leu Ser Ser
Gly Val Gln Ile Asn Val Cys Lys Asp Trp Asn 245 250 255Ile Ser Gly
Ser Ser Lys Leu Asn Ser His Phe Ala Leu Ile Val Val 260 265 270Phe
Asp Cys Leu Ile Ile Cys Phe Cys Leu Leu Ser Leu Ile Leu Cys 275 280
285Thr Arg Ser Val His Thr Gly Phe Leu Leu Gln Thr Glu Tyr Arg Arg
290 295 300Phe Met Ser Ser Gln His Ser Lys Ser Val Ser Trp Ser Glu
Arg Leu305 310 315 320Glu Phe Ile Asn Gly Trp Tyr Ile Leu Ile Ile
Ile Ser Asp Ala Leu 325 330 335Thr Ile Ala Gly Ser Ile Leu Lys Ile
Cys Ile Gln Ser Lys Glu Leu 340 345 350Thr Ser Tyr Asp Val Cys Ser
Ile Leu Leu Gly Thr Ala Thr Met Leu 355 360 365Val Trp Ile Gly Val
Met Arg Tyr Leu Ser Phe Phe Gln Lys Tyr Tyr 370 375 380Ile Leu Ile
Leu Thr Leu Lys Ala Ala Leu Pro Asn Val Ile Arg Phe385 390 395
400Ser Ile Cys Ala Val Met Ile Tyr Leu Ser Tyr Cys Phe Cys Gly Trp
405 410 415Ile Val Leu Gly Pro His His Glu Asn Phe Arg Thr Phe Ser
Arg Val 420 425 430Ala Gly Cys Leu Phe Ser Met Ile Asn Gly Asp Glu
Ile Tyr Ser Thr 435 440 445Phe Thr Lys Leu Arg Glu Tyr Ser Thr Leu
Val Trp Leu Phe Ser Arg 450 455 460Leu Tyr Val Tyr Ser Phe Ile Pro
Val Phe Thr Tyr Met Val Leu Ser465 470 475 480Val Phe Ile Ala Leu
Ile Thr Asp Thr Tyr Glu Thr Ile Arg Val Ser 485 490 495Tyr Phe Ser
Phe Ser Glu Ser Ser Cys Lys 500 505171659DNAHomo sapiens
17atggccgacc ctgaggtggt ggtgtcctcc tgctctagcc acgaggaaga gaaccggtgc
60aacttcaacc agcagaccag ccccagcgag gaactgctgc tggaagatca gatgcggcgg
120aagctgaagt tcttcttcat gaacccctgc gagaagttct gggccagagg
ccggaagcct 180tggaagctgg ccatccagat cctgaagatc gccatggtga
ccatccagct ggtgctgttc 240ggcctgagca accagatggt ggtggccttc
aaagaggaaa acacaatcgc cttcaagcac 300ctgtttctga agggctacat
ggaccggatg gacgacacct acgccgtgta cacccagagc 360gacgtgtacg
accagctgat cttcgccgtg aaccagtacc tgcagctgta caacgtgagc
420gtgggcaacc acgcctacga gaacaagggc accaagcaga gcgccatggc
catctgccag 480cacttctaca agcggggcaa catctacccc ggcaacgaca
ccttcgacat cgaccccgag 540atcgagacag agtgcttctt cgtggagccc
gacgagcctt tccacatcgg cacccctgcc 600gagaacaagc tgaacctgac
cctggacttc caccggctgc tgaccgtgga gctgcagttc 660aagctgaagg
ccatcaacct gcagaccgtg cggcaccagg aactgcccga ctgctacgac
720ttcaccctga ccatcacctt cgataacaag gcccacagcg gccggatcaa
gatcagcctg 780gacaacgaca tcagcatccg ggagtgcaag gactggcacg
tgagcggcag catccagaaa 840aacacccact acatgatgat cttcgacgcc
ttcgtgatcc tgacctgcct ggtgtccctg 900atcctgtgca tcagaagcgt
catcaggggc ctgcagctcc agcaggaatt cgtcaacttc 960ttcctgctgc
actacaagaa agaagtgtcc gtcagcgacc agatggaatt tgtgaacggc
1020tggtacatca tgatcatcat cagcgacatc ctgacaatca tcggcagcat
tctgaagatg 1080gaaatccagg ccaagagcct gaccagctac gacgtgtgca
gcattctgct gggaacctcc 1140accatgctgg tctggctcgg cgtgatcaga
tacctgggct tcttcgccaa gtacaacctg 1200ctgattctga cactgcaggc
cgccctgccc aacgtgatcc ggttctgctg ctgcgccgcc 1260atgatctacc
tgggctactg cttctgcggc tggatcgtgc tgggccccta ccacgacaag
1320ttccggtccc tgaacatggt gtccgagtgc ctgttcagcc tgatcaacgg
cgacgacatg 1380ttcgccacct tcgccaagat gcagcagaaa agctacctgg
tctggctgtt cagccggatc 1440tacctgtaca gcttcatcag cctgttcatc
tacatgatcc tgagcctgtt tatcgccctg 1500atcaccgata cctacgagac
aatcaagcag taccagcagg acggcttccc cgagacagag 1560ctgcggacct
tcatcagcga gtgtaaggac ctgcccaaca gcggcaagta ccggctggaa
1620gatgaccccc ccgtgtccct gttctgctgt tgcaagaag 1659181659DNAHomo
sapiens 18atggcagatc ctgaggtagt tgtgagtagc tgcagctctc atgaagagga
aaatcgctgc 60aattttaacc agcaaacatc tccatctgag gagcttctat tagaagacca
gatgaggcga 120aaactcaaat tttttttcat gaatccctgt
gagaagttct gggctcgagg tagaaaacca 180tggaaacttg ccatacaaat
tctaaaaatt gcaatggtga ctatccagct ggtcttattt 240gggctaagta
accagatggt ggtagctttc aaggaagaga atactatagc attcaaacac
300cttttcctaa aaggatatat ggaccgaatg gatgacacat atgcagtgta
cacacaaagt 360gacgtgtatg atcagttaat cttcgcagta aaccagtact
tgcagctata caatgtctcc 420gttgggaatc atgcttatga gaacaaaggt
accaagcaat ctgctatggc aatctgtcag 480cacttctaca agcgaggaaa
catctaccct ggaaatgata cctttgacat cgatccagaa 540attgaaactg
agtgtttctt tgtggagcca gatgaacctt ttcacattgg gacaccagca
600gaaaataaac tgaacttaac actggacttc cacagactcc taacagtgga
gcttcagttt 660aaactgaaag ccattaatct gcagacagtt cgtcatcaag
aactccctga ctgttatgac 720tttactctga ctataacatt tgacaacaag
gcccatagtg gaagaattaa aataagttta 780gataatgaca tttccatcag
agaatgtaaa gactggcatg tatctggatc aattcagaag 840aacactcatt
acatgatgat ctttgatgcc tttgtcattc tgacttgctt ggtttcatta
900atcctctgca ttagatctgt gattagagga cttcagcttc agcaggagtt
tgtcaatttt 960ttcctcctcc attataagaa ggaagtttct gtttctgatc
aaatggaatt tgtcaatgga 1020tggtacatta tgattattat tagtgacata
ttgacaatca ttggatcaat tctaaaaatg 1080gaaatccaag ctaagagtct
aactagttat gatgtctgta gcatacttct tgggacttct 1140accatgcttg
tgtggcttgg agtcatccga tacctcggtt tctttgcaaa gtacaacctc
1200ctcattttga cccttcaggc agcgctgccc aatgtcatca ggttctgctg
ctgtgcagct 1260atgatttact taggttactg cttctgtgga tggatcgtgc
tggggcctta ccatgacaag 1320tttcgttctc tgaacatggt ctctgagtgc
cttttctctc tgataaatgg agatgatatg 1380tttgccacgt ttgcaaaaat
gcagcaaaaa agttacttag tctggctgtt tagtagaatt 1440tacctctact
cattcatcag cctctttata tatatgattt taagtctttt cattgcactg
1500atcactgata catacgaaac aattaagcaa taccaacaag atggcttccc
agagactgaa 1560cttcgtacat ttatatcaga atgcaaagat ctacccaact
ctggaaaata cagattagaa 1620gatgaccctc cagtatcttt attctgctgt
tgtaaaaag 1659192000DNARattus norvegicus 19gttcgacaga agcttgtgat
ttatggtcca aggaatccag tgtcagatca atagacaaaa 60tgccccaggg aagttgtgtg
tgcattctac tggacagatc agagactggt cagaacaggt 120gcttggctgg
cggtgcgtcc aaacctcaga gatggcaaat cctgaggtgg tggtaagcag
180ctgcagttct caccaggatg aaagtccctg cactttctac ccgagctcat
cccagtccga 240gcagcttctc ttagaagatc agatgaggcg gaaactcaaa
ttctttttta tgaatccttg 300cgagaagttc tgggctcggg gtaggaagcc
atggaaactt gccatacaga ttctgaaaat 360cgctatggtg actatccagc
tggttctgtt tggactaagt aaccagatgg tagtagcttt 420caaggaagag
aacacgatag ccttcaaaca cctcttcctg aaaggctaca tggaccgaat
480ggacgacacc tacgcggtgt acactcagaa tgatgtgtac gaccagatca
tctttgcagt 540gacccggtac ttgcaacttc gaaacatctc cgtcggcaac
catgcttatg agaacaaggg 600gactaagcag tcagcaatgg cagtctgtca
gcacttctac aggcaaggca ccatctgccc 660cgggaacgat accttcgaca
tcgatccaga agtcgaaaca gactgtttcc ttatagagcc 720agaggaagct
ttccacatgg gaacacctgg agaaaacaaa ctcaacctga ccctggactt
780ccacagactt ctgacagtgg agctccaatt taagctcaaa gccatcaacc
tgcagacagt 840tcgccaccag gagcttcctg actgttacga ctttaccctg
actataacat tcgacaacaa 900ggcacacagt ggaagaatca aaataagttt
agacaacgac atttctatca gagaatgcaa 960agattggcac gtgtctggat
caattcagaa gaacacccac tacatgatga tcttcgatgc 1020ctttgttatc
ctgacctgct tgtcctcgct ggtgctctgc gccaggtctg tgattcgggg
1080tcttcagctt cagcaggagt ttgtcaactt tttccttctt cactacaaga
aggaagtttc 1140ggcctctgat cagatggagt tcatcaacgg gtggtacatt
atgatcatcg ttagtgacat 1200actgacgatc gttggatcga ttctgaaaat
ggaaatccaa gccaagagtc ttacaagcta 1260cgatgtctgt agcatacttc
ttgggacttc caccatgctc gtgtggcttg gcgttatccg 1320atacctgggt
ttctttgcga agtacaatct ccttatcctg accctccagg cagcgctgcc
1380caatgtcatc aggttctgtt gctgtgcggc tatgatctat cttgggtatt
gcttttgcgg 1440atggattgtg ctgggccctt accatgagaa gttccgctct
ctgaacaagg tctctgagtg 1500cctattctca ctgataaatg gagacgacat
gttttccacg ttcgcgaaaa tgcagcagaa 1560aagttacctg gtgtggctgt
tcagccgcgt ctacctgtac tcgttcatca gcctcttcat 1620ctacatgatc
ttgagccttt tcatcgcgct catcacagac acgtacgaaa ccattaagca
1680ctaccagcaa gatggcttcc cagagacgga acttcgaaag tttatcgctg
aatgcaaaga 1740cctccccaac tctggaaaat acagattgga agatgaccct
ccaggttctt tattctgctg 1800ctgcaaaaag taactgtcgg gtttctctgt
gcctgggagg aaaatacagt gtgtggatga 1860gtcagagaca atatggatta
ttggtaatca cgcaacagtg tgttcagata ctagtgttct 1920gagttaactc
acagctatga ctttgcgggg cctgttaaat atatttttaa atattaaaaa
1980aaaaaaaaaa aaaaaaaaaa 200020553PRTRattus norvegicus 20Met Ala
Asn Pro Glu Val Val Val Ser Ser Cys Ser Ser His Gln Asp1 5 10 15Glu
Ser Pro Cys Thr Phe Tyr Pro Ser Ser Ser Gln Ser Glu Gln Leu 20 25
30Leu Leu Glu Asp Gln Met Arg Arg Lys Leu Lys Phe Phe Phe Met Asn
35 40 45Pro Cys Glu Lys Phe Trp Ala Arg Gly Arg Lys Pro Trp Lys Leu
Ala 50 55 60Ile Gln Ile Leu Lys Ile Ala Met Val Thr Ile Gln Leu Val
Leu Phe65 70 75 80Gly Leu Ser Asn Gln Met Val Val Ala Phe Lys Glu
Glu Asn Thr Ile 85 90 95Ala Phe Lys His Leu Phe Leu Lys Gly Tyr Met
Asp Arg Met Asp Asp 100 105 110Thr Tyr Ala Val Tyr Thr Gln Asn Asp
Val Tyr Asp Gln Ile Ile Phe 115 120 125Ala Val Thr Arg Tyr Leu Gln
Leu Arg Asn Ile Ser Val Gly Asn His 130 135 140Ala Tyr Glu Asn Lys
Gly Thr Lys Gln Ser Ala Met Ala Val Cys Gln145 150 155 160His Phe
Tyr Arg Gln Gly Thr Ile Cys Pro Gly Asn Asp Thr Phe Asp 165 170
175Ile Asp Pro Glu Val Glu Thr Asp Cys Phe Leu Ile Glu Pro Glu Glu
180 185 190Ala Phe His Met Gly Thr Pro Gly Glu Asn Lys Leu Asn Leu
Thr Leu 195 200 205Asp Phe His Arg Leu Leu Thr Val Glu Leu Gln Phe
Lys Leu Lys Ala 210 215 220Ile Asn Leu Gln Thr Val Arg His Gln Glu
Leu Pro Asp Cys Tyr Asp225 230 235 240Phe Thr Leu Thr Ile Thr Phe
Asp Asn Lys Ala His Ser Gly Arg Ile 245 250 255Lys Ile Ser Leu Asp
Asn Asp Ile Ser Ile Arg Glu Cys Lys Asp Trp 260 265 270His Val Ser
Gly Ser Ile Gln Lys Asn Thr His Tyr Met Met Ile Phe 275 280 285Asp
Ala Phe Val Ile Leu Thr Cys Leu Ser Ser Leu Val Leu Cys Ala 290 295
300Arg Ser Val Ile Arg Gly Leu Gln Leu Gln Gln Glu Phe Val Asn
Phe305 310 315 320Phe Leu Leu His Tyr Lys Lys Glu Val Ser Ala Ser
Asp Gln Met Glu 325 330 335Phe Ile Asn Gly Trp Tyr Ile Met Ile Ile
Val Ser Asp Ile Leu Thr 340 345 350Ile Val Gly Ser Ile Leu Lys Met
Glu Ile Gln Ala Lys Ser Leu Thr 355 360 365Ser Tyr Asp Val Cys Ser
Ile Leu Leu Gly Thr Ser Thr Met Leu Val 370 375 380Trp Leu Gly Val
Ile Arg Tyr Leu Gly Phe Phe Ala Lys Tyr Asn Leu385 390 395 400Leu
Ile Leu Thr Leu Gln Ala Ala Leu Pro Asn Val Ile Arg Phe Cys 405 410
415Cys Cys Ala Ala Met Ile Tyr Leu Gly Tyr Cys Phe Cys Gly Trp Ile
420 425 430Val Leu Gly Pro Tyr His Glu Lys Phe Arg Ser Leu Asn Lys
Val Ser 435 440 445Glu Cys Leu Phe Ser Leu Ile Asn Gly Asp Asp Met
Phe Ser Thr Phe 450 455 460Ala Lys Met Gln Gln Lys Ser Tyr Leu Val
Trp Leu Phe Ser Arg Val465 470 475 480Tyr Leu Tyr Ser Phe Ile Ser
Leu Phe Ile Tyr Met Ile Leu Ser Leu 485 490 495Phe Ile Ala Leu Ile
Thr Asp Thr Tyr Glu Thr Ile Lys His Tyr Gln 500 505 510Gln Asp Gly
Phe Pro Glu Thr Glu Leu Arg Lys Phe Ile Ala Glu Cys 515 520 525Lys
Asp Leu Pro Asn Ser Gly Lys Tyr Arg Leu Glu Asp Asp Pro Pro 530 535
540Gly Ser Leu Phe Cys Cys Cys Lys Lys545 55021751DNASus scrofa
21gtgaaatggc agatcctgag cctgtcataa gtagctgcag ctctcgtgaa gaggaaaatc
60gctgcacttt taaccagcac acatgtccct ctgaggagcg tctattagaa gaccagatga
120ggcgaaaact caaatttttt ttcatgactc cttgtgagaa gttctggact
cgaggtcgaa 180aaccatggaa acttgccatg caagttctaa aaattgcgat
ggtgactatc cagctgatct 240ttttcgggct aagtaaccag atggtggtag
ctttcaagga agagaacacg atagcattta 300aacacctctt tctaaagggc
tatgtggacc agatggatga cacatatgcc gtgtacaccc 360aaagcgacgt
atacgatcgg atcgtcttcg cagtgaacca gtacttgcag ctacgcagca
420tctcggttgg gaaccacgct tacgagaaca agggcgcgga gcagtcggcc
atggcgatct 480gttggcactt ctacaagcaa ggaaacatct gtcctggaaa
tgacaccttt gacgttgatc 540cagaagtaaa aactgaatgt ttctttgttg
agccggatga agctgttgac actggaacac 600tggaggagaa taagctcaac
ttaacccttg actttcacag actcctaacg gtggagctgc 660agtttaaact
caaggccatt aatctgcaga cgattcgcca tcacgaactc cctgactgtt
720atgacttcac cctgaccata acatttgaca a 75122248PRTSus scrofa 22Met
Ala Asp Pro Glu Pro Val Ile Ser Ser Cys Ser Ser Arg Glu Glu1 5 10
15Glu Asn Arg Cys Thr Phe Asn Gln His Thr Cys Pro Ser Glu Glu Arg
20 25 30Leu Leu Glu Asp Gln Met Arg Arg Lys Leu Lys Phe Phe Phe Met
Thr 35 40 45Pro Cys Glu Lys Phe Trp Thr Arg Gly Arg Lys Pro Trp Lys
Leu Ala 50 55 60Met Gln Val Leu Lys Ile Ala Met Val Thr Ile Gln Leu
Ile Phe Phe65 70 75 80Gly Leu Ser Asn Gln Met Val Val Ala Phe Lys
Glu Glu Asn Thr Ile 85 90 95Ala Phe Lys His Leu Phe Leu Lys Gly Tyr
Val Asp Gln Met Asp Asp 100 105 110Thr Tyr Ala Val Tyr Thr Gln Ser
Asp Val Tyr Asp Arg Ile Val Phe 115 120 125Ala Val Asn Gln Tyr Leu
Gln Leu Arg Ser Ile Ser Val Gly Asn His 130 135 140Ala Tyr Glu Asn
Lys Gly Ala Glu Gln Ser Ala Met Ala Ile Cys Trp145 150 155 160His
Phe Tyr Lys Gln Gly Asn Ile Cys Pro Gly Asn Asp Thr Phe Asp 165 170
175Val Asp Pro Glu Val Lys Thr Glu Cys Phe Phe Val Glu Pro Asp Glu
180 185 190Ala Val Asp Thr Gly Thr Leu Glu Glu Asn Lys Leu Asn Leu
Thr Leu 195 200 205Asp Phe His Arg Leu Leu Thr Val Glu Leu Gln Phe
Lys Leu Lys Ala 210 215 220Ile Asn Leu Gln Thr Ile Arg His His Glu
Leu Pro Asp Cys Tyr Asp225 230 235 240Phe Thr Leu Thr Ile Thr Phe
Asp 245232880DNAPan troglodytes 23cttactccaa tcaagcctct gcccgccagg
aataggtaac ctgtgtgtgt ccgtttgctc 60cttctaagag catgcctgat agatacttcg
gtagcctctc cggatggccc cttcgtcggg 120tagcctctcc tgatggggtc
cttcgcccac cctgcctccc gcgccggcgc tccgggtgaa 180tgtcaagggt
ggctggctgc gaatacctcc ttcagctgct ggggttcccg acagtttgca
240gtttttaaaa gtgcaccctc ggaagggctt ttcagactgg gtaaagctga
cttttccaag 300agatggcaga tcctgaggta gttgtgagta gctgcagctc
tcatgaagag gaaaatcgct 360gcaattttaa ccagcaaaca tctccatctg
aggagcttct attagaagac cagatgaggc 420gaaaactcaa attttttttc
atgaatccct gtgagaagtt ctgggctcga ggtagaaaac 480catggaaact
tgccatacaa attctaaaaa ttgcaatggt gactatccag ctggtcttat
540ttgggctaag taaccagatg gtggtagctt tcaaggaaga gaatactata
gcattcaaac 600accttttcct aaaaggatat atggaccgaa tggatgacac
atatgcagtg tacacacaaa 660gtgacgtgta tgatcagtta atcttcgcag
taaaccagta cttgcagcta tacaatgtct 720ccgttgggaa tcatgcttat
gagaacaaag gtaccaagca atctgctatg gcaatctgtc 780agcacttcta
caagcgagga aacatctacc ctggaaatga tacctttgac atcgatccag
840aaattgaaac tgagtgtttc tttgtggagc cagatgaacc ttttcacatt
gggacaccag 900cagaaaataa actgaactta acactggact tccacagact
cctaacagtg gagcttcagt 960ttaaactgaa agccattaat ctgcagacag
ttcgtcatca agaactccct gactgttatg 1020actttactct gactataaca
tttgacaaca aggcccatag tggaagaatt aaaataagtt 1080tagataatga
catttccatc agagaatgta aagactggca tgtatctgga tcaattcaga
1140agaacactca ttacatgatg atctttgatg cctttgtcat tctgacttgc
ttggtttcat 1200taatcctctg cattagatct gtgattagag gacttcagct
tcagcaggta gggaacgttg 1260ctttctagga atgctactga cattttgatt
gacagagaca ttcactgtgc ctcccctctt 1320ttccctaaag gagtttgtca
attttttcct cctccattat aagaagggag tttctgtttc 1380tgatcaaatg
gaatttgtca atggatggta cattatgatt attattagtg acatattgac
1440aatcattgga tcaattctaa aaatggaaat ccaagctaag gtaatttttt
tcctaatcat 1500gctattgtta gtgtcagatt tgcactaatg gtaatgtatt
tttccagaat gtaagaattt 1560tcagaatgaa ttgtttcttc caaactgtat
atcaagtaga cttgaaattg gtaatggtaa 1620ttttcttaaa tctagtcagg
aggtctctta ggcagagttt ttcaaagtgt gatccacaaa 1680ccattgcatc
agaatcattg ggtgcctggt aaagtgtacc atgttagacc tactgaattc
1740agactcttcg gcggggcctg tgaattctta cacacaccaa aattcataca
caaccaaggt 1800aactaaggta agagtttttt ttttttttaa atcttacaag
aaatgctcaa atctttaaca 1860aaaatgagtg ggtctatagg ggaaagtgag
gtcaaggcac tatggtgtgc atgcttgcat 1920ttgtttcctc cgtccattca
aagtgagaat gctcccattt tcttacttta ccattgatgt 1980gctacaagct
tatttatttt aagactaacc tagcctaaaa atcaactgtc cccacaaaat
2040aaaaatcaca ttaaaaaaac taatagtgtt cagactaatc ttgctcaaac
ttatgtttcc 2100ctagtcttga tgcgactgat tgagtcacct ggggagttgg
ttataaacct gggcagagac 2160cccaaatgca atggctcaga gaagatagga
gcttatttct gtcttatgca atagtcagaa 2220tgggttttac agactggtga
gtagctcaac atctcacagt cattcaggca cccatgttcc 2280tcccattttg
tttctctgcc atcccttaag gacttgccct gactgcatga ttattgctgt
2340gttgcctcaa acaggttgca gcttatggga agcaaaaaca cggtatggtg
gaagctctcc 2400catagactga tggcttggct caagagtggc cgactttatt
tctgtacata tcccactgga 2460tagaatttag tcaatcctaa ctgcagaggg
agccagggaa cacagcccag gcatgtgcct 2520aggaagggga gaatgggttt
aggttgacac ttagcagctg ccactatatg tggctatagt 2580atgtatcatt
ggaatagatg tttaacttta gggacaaata aacaaaaaac caaaacaaaa
2640aaaggagtaa ggggagagat ttgcagcaaa tctttatttt taccaacctc
aactatcatt 2700aatttcagtg aaccctaaat ggtgtccaac aaaatatctt
tctagaccat tcaccgtctc 2760tgcctcatag atgatcatat catgttttct
tctcttctga aacctctaat acccttgtcc 2820tatcctcatt ctaagctgat
gaccttactt cctatttcac aaaaataata gaaaaaaaaa 288024321PRTPan
troglodytes 24Met Ala Asp Pro Glu Val Val Val Ser Ser Cys Ser Ser
His Glu Glu1 5 10 15Glu Asn Arg Cys Asn Phe Asn Gln Gln Thr Ser Pro
Ser Glu Glu Leu 20 25 30Leu Leu Glu Asp Gln Met Arg Arg Lys Leu Lys
Phe Phe Phe Met Asn 35 40 45Pro Cys Glu Lys Phe Trp Ala Arg Gly Arg
Lys Pro Trp Lys Leu Ala 50 55 60Ile Gln Ile Leu Lys Ile Ala Met Val
Thr Ile Gln Leu Val Leu Phe65 70 75 80Gly Leu Ser Asn Gln Met Val
Val Ala Phe Lys Glu Glu Asn Thr Ile 85 90 95Ala Phe Lys His Leu Phe
Leu Lys Gly Tyr Met Asp Arg Met Asp Asp 100 105 110Thr Tyr Ala Val
Tyr Thr Gln Ser Asp Val Tyr Asp Gln Leu Ile Phe 115 120 125Ala Val
Asn Gln Tyr Leu Gln Leu Tyr Asn Val Ser Val Gly Asn His 130 135
140Ala Tyr Glu Asn Lys Gly Thr Lys Gln Ser Ala Met Ala Ile Cys
Gln145 150 155 160His Phe Tyr Lys Arg Gly Asn Ile Tyr Pro Gly Asn
Asp Thr Phe Asp 165 170 175Ile Asp Pro Glu Ile Glu Thr Glu Cys Phe
Phe Val Glu Pro Asp Glu 180 185 190Pro Phe His Ile Gly Thr Pro Ala
Glu Asn Lys Leu Asn Leu Thr Leu 195 200 205Asp Phe His Arg Leu Leu
Thr Val Glu Leu Gln Phe Lys Leu Lys Ala 210 215 220Ile Asn Leu Gln
Thr Val Arg His Gln Glu Leu Pro Asp Cys Tyr Asp225 230 235 240Phe
Thr Leu Thr Ile Thr Phe Asp Asn Lys Ala His Ser Gly Arg Ile 245 250
255Lys Ile Ser Leu Asp Asn Asp Ile Ser Ile Arg Glu Cys Lys Asp Trp
260 265 270His Val Ser Gly Ser Ile Gln Lys Asn Thr His Tyr Met Met
Ile Phe 275 280 285Asp Ala Phe Val Ile Leu Thr Cys Leu Val Ser Leu
Ile Leu Cys Ile 290 295 300Arg Ser Val Ile Arg Gly Leu Gln Leu Gln
Gln Val Gly Asn Val Ala305 310 315 320Phe251868DNAPan troglodytes
25cctctagaga tggcagatcc tgaggtagtt gtgagtagct gcagctctca tgaagaggaa
60aatcgctgca attttaacca gcaaacatct ccatctgagg agcttctatt agaagaccag
120atgaggcgaa aactcaaatt ttttttcatg aatccctgtg agaagttctg
ggctcgaggt 180agaaaaccat ggaaacttgc catacaaatt ctaaaaattg
caatggtgac tatccagctg 240gtcttatttg ggctaagtaa ccagatggtg
gtagctttca aggaagagaa tactatagca 300ttcaaacacc ttttcctaaa
aggatatatg gaccgaatgg atgacacata tgcagtgtac 360acacaaagtg
acgtgtatga tcagttaatc ttcgcagtaa accagtactt gcagctatac
420aatgtctccg ttgggaatca tgcttatgag aacaaaggta ccaagcaatc
tgctatggca 480atctgtcagc acttctacaa gcgaggaaac atctaccctg
gaaatgatac ctttgacatc 540gatccagaaa ttgaaactga gtgtttcttt
gtggagccag atgaaccttt tcacattggg 600acaccagcag aaaataaact
gaacttaaca ctggacttcc acagactcct aacagtggag 660cttcagttta
aactgaaagc cattaatctg cagacagttc gtcatcaaga actccctgac
720tgttatgact ttactctgac tataacattt gacaacaagg cccatagtgg
aagaattaaa 780ataagtttag ataatgacat ttccatcaga gaatgtaaag
actggcattc tccctccgtc 840gcccagcctg gaaacactca ttacatgatg
atctttgatg cctttgtcat tctgacttgc 900ttggtttcat taatcctctg
cattagatct gtgattagag gacttcagct tcagcaggag 960tttgtcaatt
ttttcctcct ccattataag aagggagttt ctgtttctga tcaaatggaa
1020tttgtcaatg gatggtacat tatgattatt attagtgaca tattgacaat
cattggatca 1080attctaaaaa tggaaatcca agctaagagt ctaactagtt
atgatgtctg tagcatactt 1140cttgggactt ctaccatgct tgtgtggctt
ggagtcatcc gatacctcgg tttctttgca 1200aagtacaatc tcctcatttt
gacccttcag gcagcactgc ccaatgtcat caggttctgc 1260tgctgtgcag
ctatgattta cttaggttac tgcttctgtg gatggatcgt gctggggcct
1320taccatgaca agtttcgttc tctgaacatg gtctctgagt gccttttctc
tctgataaat 1380ggagatgata tgtttgccac gtttgcaaaa atgcagcaaa
aaagttactt agtctggctg 1440tttagtagaa tttacctcta ctcattcatc
agcctcttta tatatatgat tttaagtctt 1500ttcattgcac tgatcactga
tacatacgaa acaattaagc aataccaaca agatggcttc 1560ccagagactg
aacttcgtac atttatatca gaatgcaaag atctacccaa ctctggaaaa
1620tacagattag aagatgaccc tccagtatct ttattctgct gttgtaaaaa
gtagctatca 1680ggtttatctg tactttagag gaaaatataa tgtgtagctg
agttagaaca ctgtggatat 1740tctgagatca gatgtagtat gtttgaagac
tgttattttg agctaattga gacctataat 1800tcaccaataa ctgtttatat
ttttaaaagc aatatttaat gtctttgcag ctttatgctg 1860ggcttgtt
186826554PRTPan troglodytes 26Met Ala Asp Pro Glu Val Val Val Ser
Ser Cys Ser Ser His Glu Glu1 5 10 15Glu Asn Arg Cys Asn Phe Asn Gln
Gln Thr Ser Pro Ser Glu Glu Leu 20 25 30Leu Leu Glu Asp Gln Met Arg
Arg Lys Leu Lys Phe Phe Phe Met Asn 35 40 45Pro Cys Glu Lys Phe Trp
Ala Arg Gly Arg Lys Pro Trp Lys Leu Ala 50 55 60Ile Gln Ile Leu Lys
Ile Ala Met Val Thr Ile Gln Leu Val Leu Phe65 70 75 80Gly Leu Ser
Asn Gln Met Val Val Ala Phe Lys Glu Glu Asn Thr Ile 85 90 95Ala Phe
Lys His Leu Phe Leu Lys Gly Tyr Met Asp Arg Met Asp Asp 100 105
110Thr Tyr Ala Val Tyr Thr Gln Ser Asp Val Tyr Asp Gln Leu Ile Phe
115 120 125Ala Val Asn Gln Tyr Leu Gln Leu Tyr Asn Val Ser Val Gly
Asn His 130 135 140Ala Tyr Glu Asn Lys Gly Thr Lys Gln Ser Ala Met
Ala Ile Cys Gln145 150 155 160His Phe Tyr Lys Arg Gly Asn Ile Tyr
Pro Gly Asn Asp Thr Phe Asp 165 170 175Ile Asp Pro Glu Ile Glu Thr
Glu Cys Phe Phe Val Glu Pro Asp Glu 180 185 190Pro Phe His Ile Gly
Thr Pro Ala Glu Asn Lys Leu Asn Leu Thr Leu 195 200 205Asp Phe His
Arg Leu Leu Thr Val Glu Leu Gln Phe Lys Leu Lys Ala 210 215 220Ile
Asn Leu Gln Thr Val Arg His Gln Glu Leu Pro Asp Cys Tyr Asp225 230
235 240Phe Thr Leu Thr Ile Thr Phe Asp Asn Lys Ala His Ser Gly Arg
Ile 245 250 255Lys Ile Ser Leu Asp Asn Asp Ile Ser Ile Arg Glu Cys
Lys Asp Trp 260 265 270His Ser Pro Ser Val Ala Gln Pro Gly Asn Thr
His Tyr Met Met Ile 275 280 285Phe Asp Ala Phe Val Ile Leu Thr Cys
Leu Val Ser Leu Ile Leu Cys 290 295 300Ile Arg Ser Val Ile Arg Gly
Leu Gln Leu Gln Gln Glu Phe Val Asn305 310 315 320Phe Phe Leu Leu
His Tyr Lys Lys Gly Val Ser Val Ser Asp Gln Met 325 330 335Glu Phe
Val Asn Gly Trp Tyr Ile Met Ile Ile Ile Ser Asp Ile Leu 340 345
350Thr Ile Ile Gly Ser Ile Leu Lys Met Glu Ile Gln Ala Lys Ser Leu
355 360 365Thr Ser Tyr Asp Val Cys Ser Ile Leu Leu Gly Thr Ser Thr
Met Leu 370 375 380Val Trp Leu Gly Val Ile Arg Tyr Leu Gly Phe Phe
Ala Lys Tyr Asn385 390 395 400Leu Leu Ile Leu Thr Leu Gln Ala Ala
Leu Pro Asn Val Ile Arg Phe 405 410 415Cys Cys Cys Ala Ala Met Ile
Tyr Leu Gly Tyr Cys Phe Cys Gly Trp 420 425 430Ile Val Leu Gly Pro
Tyr His Asp Lys Phe Arg Ser Leu Asn Met Val 435 440 445Ser Glu Cys
Leu Phe Ser Leu Ile Asn Gly Asp Asp Met Phe Ala Thr 450 455 460Phe
Ala Lys Met Gln Gln Lys Ser Tyr Leu Val Trp Leu Phe Ser Arg465 470
475 480Ile Tyr Leu Tyr Ser Phe Ile Ser Leu Phe Ile Tyr Met Ile Leu
Ser 485 490 495Leu Phe Ile Ala Leu Ile Thr Asp Thr Tyr Glu Thr Ile
Lys Gln Tyr 500 505 510Gln Gln Asp Gly Phe Pro Glu Thr Glu Leu Arg
Thr Phe Ile Ser Glu 515 520 525Cys Lys Asp Leu Pro Asn Ser Gly Lys
Tyr Arg Leu Glu Asp Asp Pro 530 535 540Pro Val Ser Leu Phe Cys Cys
Cys Lys Lys545 550272546DNAPan troglodytes 27tcctctagag atggcagatc
ctgaggtagt tgtgagtagc tgcagctctc atgaagagga 60aaatcgctgc aattttaacc
agcaaacatc tccatctgag gagcttctat tagaagacca 120gatgaggcga
aaactcaaat tttttttcat gaatccctgt gagaagttct gggctcgagg
180tagaaaacca tggaaacttg ccatacaaat tctaaaaatt gcaatggtga
ctatccagta 240cttgcagcta tacaatgtct ccgttgggaa tcatgcttat
gagaacaaag gtaccaagca 300atctgctatg gcaatctgtc agcacttcta
caagcgagga aacatctacc ctggaaatga 360tacctttgac atcgatccag
aaattgaaac tgagtgtttc tttgtggagc cagatgaacc 420ttttcacatt
gggacaccag cagaaaataa actgaactta acactggact tccacagact
480cctaacagtg gagcttcagt ttaaactgaa agccattaat ctgcagacag
ttcgtcatca 540agaactccct gactgttatg actttactct gactataaca
tttgacaaca aggcccatag 600tggaagaatt aaaataagtt tagataatga
catttccatc agagaatgta aagactggca 660tgtatctgga tcaattcaga
agaacactca ttacatgatg atctttgatg cctttgtcat 720tctgacttgc
ttggtttcat taatcctctg cattagatct gtgattagag gacttcagct
780tcagcaggag tttgtcaatt ttttcctcct ccattataag aagggagttt
ctgtttctga 840tcaaatggaa tttgtcaatg gatggtacat tatgattatt
attagtgaca tattgacaat 900cattggatca attctaaaaa tggaaatcca
agctaagagt ctaactagtt atgatgtctg 960tagcatactt cttgggactt
ctaccatgct tgtgtggctt ggagtcatcc gatacctcgg 1020tttctttgca
aagtacaatc tcctcatttt gacccttcag gcagcactgc ccaatgtcat
1080caggttctgc tgctgtgcag ctatgattta cttaggttac tgcttctgtg
gatggatcgt 1140gctggggcct taccatgaca agtttcgttc tctgaacatg
gtctctgagt gccttttctc 1200tctgataaat ggagatgata tgtttgccac
gtttgcaaaa atgcagcaaa aaagttactt 1260agtctggctg tttagtagaa
tttacctcta ctcattcatc agcctcttta tatatatgat 1320tttaagtctt
ttcattgcac tgatcactga tacatacgaa acaattaagc aataccaaca
1380agatggcttc ccagagactg aacttcgtac atttatatca gaatgcaaag
atctacccaa 1440ctctggaaaa tacagattag aagatgaccc tccagtatct
ttattctgct gttgtaaaaa 1500gtagctatca ggtttatctg tactttagag
gaaaatataa tgtgtagctg agttagaaca 1560ctgtggatat tctgagatca
gatgtagtat gtttgaagac tgttattttg agctaattga 1620gacctataat
tcaccaataa ctgtttatat ttttaaaagc aatatttaat gtctttgcag
1680ctttatgctg ggcttgtttt taaaacaact ttaatgagga aagctattgg
attattatta 1740tttcttgttt attttgccat ggctttagaa tgtattctgt
atgcctctct tttgctctga 1800tactcttgct tcctgctatt ctgattgtgc
agactgtgta attagtggaa aacaatcctt 1860ggtctgactg tgactttgga
caactcagta accctggctt ggaccactct caggagtcca 1920tccttgagag
agtgggtgta gttaccattt atacagtaat cattgcattt taaaatctgc
1980tcttgaaagg aagaataaga gtgcaccaga ataagagcgc accagaataa
gagcgcacca 2040gctaacaatg tgatacggcc atatgtcact taaggataga
gatatgttct gagaaatgtg 2100tcattaggcg attttgtcat taaacatcat
agcatgtact tccacaaacc tagatggtat 2160agcctactac acacctaggc
tatttggtat agcctgttga tcctggggta caaatctgta 2220caacatgtta
ctgtattgaa tacagtaggc aattgtaaca caatggtaag tatctaaaca
2280tagaaaaggg acagtaaaaa tatggtttta taatcttctg ggaccaccat
tgtatatgcg 2340gtacatcgtt gaccaaaaca tcgttatcca gcatatgact
gtatttggtt atgaaagcca 2400actgttactt gattctgctt ttagttctta
agaggatcag gtttttaaat actcatttac 2460aagttttcta tcctccttca
gtgttaaagt agaaagtaaa aagagtatct tatacatgca 2520tgaaattaaa
gcatatacca aatgca 254628497PRTPan troglodytes 28Met Ala Asp Pro Glu
Val Val Val Ser Ser Cys Ser Ser His Glu Glu1 5 10 15Glu Asn Arg Cys
Asn Phe Asn Gln Gln Thr Ser Pro Ser Glu Glu Leu 20 25 30Leu Leu Glu
Asp Gln Met Arg Arg Lys Leu Lys Phe Phe Phe Met Asn 35 40 45Pro Cys
Glu Lys Phe Trp Ala Arg Gly Arg Lys Pro Trp Lys Leu Ala 50 55 60Ile
Gln Ile Leu Lys Ile Ala Met Val Thr Ile Gln Tyr Leu Gln Leu65 70 75
80Tyr Asn Val Ser Val Gly Asn His Ala Tyr Glu Asn Lys Gly Thr Lys
85 90 95Gln Ser Ala Met Ala Ile Cys Gln His Phe Tyr Lys Arg Gly Asn
Ile 100 105 110Tyr Pro Gly Asn Asp Thr Phe Asp Ile Asp Pro Glu Ile
Glu Thr Glu 115 120 125Cys Phe Phe Val Glu Pro Asp Glu Pro Phe His
Ile Gly Thr Pro Ala 130 135 140Glu Asn Lys Leu Asn Leu Thr Leu Asp
Phe His Arg Leu Leu Thr Val145 150 155 160Glu Leu Gln Phe Lys Leu
Lys Ala Ile Asn Leu Gln Thr Val Arg His 165 170 175Gln Glu Leu Pro
Asp Cys Tyr Asp Phe Thr Leu Thr Ile Thr Phe Asp 180 185 190Asn Lys
Ala His Ser Gly Arg Ile Lys Ile Ser Leu Asp Asn Asp Ile 195 200
205Ser Ile Arg Glu Cys Lys Asp Trp His Val Ser Gly Ser Ile Gln Lys
210 215 220Asn Thr His Tyr Met Met Ile Phe Asp Ala Phe Val Ile Leu
Thr Cys225 230 235 240Leu Val Ser Leu Ile Leu Cys Ile Arg Ser Val
Ile Arg Gly Leu Gln 245 250 255Leu Gln Gln Glu Phe Val Asn Phe Phe
Leu Leu His Tyr Lys Lys Gly 260 265 270Val Ser Val Ser Asp Gln Met
Glu Phe Val Asn Gly Trp Tyr Ile Met 275 280 285Ile Ile Ile Ser Asp
Ile Leu Thr Ile Ile Gly Ser Ile Leu Lys Met 290 295 300Glu Ile Gln
Ala Lys Ser Leu Thr Ser Tyr Asp Val Cys Ser Ile Leu305 310 315
320Leu Gly Thr Ser Thr Met Leu Val Trp Leu Gly Val Ile Arg Tyr Leu
325 330 335Gly Phe Phe Ala Lys Tyr Asn Leu Leu Ile Leu Thr Leu Gln
Ala Ala 340 345 350Leu Pro Asn Val Ile Arg Phe Cys Cys Cys Ala Ala
Met Ile Tyr Leu 355 360 365Gly Tyr Cys Phe Cys Gly Trp Ile Val Leu
Gly Pro Tyr His Asp Lys 370 375 380Phe Arg Ser Leu Asn Met Val Ser
Glu Cys Leu Phe Ser Leu Ile Asn385 390 395 400Gly Asp Asp Met Phe
Ala Thr Phe Ala Lys Met Gln Gln Lys Ser Tyr 405 410 415Leu Val Trp
Leu Phe Ser Arg Ile Tyr Leu Tyr Ser Phe Ile Ser Leu 420 425 430Phe
Ile Tyr Met Ile Leu Ser Leu Phe Ile Ala Leu Ile Thr Asp Thr 435 440
445Tyr Glu Thr Ile Lys Gln Tyr Gln Gln Asp Gly Phe Pro Glu Thr Glu
450 455 460Leu Arg Thr Phe Ile Ser Glu Cys Lys Asp Leu Pro Asn Ser
Gly Lys465 470 475 480Tyr Arg Leu Glu Asp Asp Pro Pro Val Ser Leu
Phe Cys Cys Cys Lys 485 490 495Lys 292714DNAPan troglodytes
29tcctctagag atggcagatc ctgaggtagt tgtgagtagc tgcagctctc atgaagagga
60aaatcgctgc aattttaacc agcaaacatc tccatctgag gagcttctat tagaagacca
120gatgaggcga aaactcaaat tttttttcat gaatccctgt gagaagttct
gggctcgagg 180tagaaaacca tggaaacttg ccatacaaat tctaaaaatt
gcaatggtga ctatccagct 240ggtcttattt gggctaagta accagatggt
ggtagctttc aaggaagaga atactatagc 300attcaaacac cttttcctaa
aaggatatat ggaccgaatg gatgacacat atgcagtgta 360cacacaaagt
gacgtgtatg atcagttaat cttcgcagta aaccagtact tgcagctata
420caatgtctcc gttgggaatc atgcttatga gaacaaaggt accaagcaat
ctgctatggc 480aatctgtcag cacttctaca agcgaggaaa catctaccct
ggaaatgata cctttgacat 540cgatccagaa attgaaactg agtgtttctt
tgtggagcca gatgaacctt ttcacattgg 600gacaccagca gaaaataaac
tgaacttaac actggacttc cacagactcc taacagtgga 660gcttcagttt
aaactgaaag ccattaatct gcagacagtt cgtcatcaag aactccctga
720ctgttatgac tttactctga ctataacatt tgacaacaag gcccatagtg
gaagaattaa 780aataagttta gataatgaca tttccatcag agaatgtaaa
gactggcatg tatctggatc 840aattcagaag aacactcatt acatgatgat
ctttgatgcc tttgtcattc tgacttgctt 900ggtttcatta atcctctgca
ttagatctgt gattagagga cttcagcttc agcaggagtt 960tgtcaatttt
ttcctcctcc attataagaa gggagtttct gtttctgatc aaatggaatt
1020tgtcaatgga tggtacatta tgattattat tagtgacata ttgacaatca
ttggatcaat 1080tctaaaaatg gaaatccaag ctaagagtct aactagttat
gatgtctgta gcatacttct 1140tgggacttct accatgcttg tgtggcttgg
agtcatccga tacctcggtt tctttgcaaa 1200gtacaatctc ctcattttga
cccttcaggc agcactgccc aatgtcatca ggttctgctg 1260ctgtgcagct
atgatttact taggttactg cttctgtgga tggatcgtgc tggggcctta
1320ccatgacaag tttcgttctc tgaacatggt ctctgagtgc cttttctctc
tgataaatgg 1380agatgatatg tttgccacgt ttgcaaaaat gcagcaaaaa
agttacttag tctggctgtt 1440tagtagaatt tacctctact cattcatcag
cctctttata tatatgattt taagtctttt 1500cattgcactg atcactgata
catacgaaac aattaagcaa taccaacaag atggcttccc 1560agagactgaa
cttcgtacat ttatatcaga atgcaaagat ctacccaact ctggaaaata
1620cagattagaa gatgaccctc cagtatcttt attctgctgt tgtaaaaagt
agctatcagg 1680tttatctgta ctttagagga aaatataatg tgtagctgag
ttagaacact gtggatattc 1740tgagatcaga tgtagtatgt ttgaagactg
ttattttgag ctaattgaga cctataattc 1800accaataact gtttatattt
ttaaaagcaa tatttaatgt ctttgcagct ttatgctggg 1860cttgttttta
aaacaacttt aatgaggaaa gctattggat tattattatt tcttgtttat
1920tttgccatgg ctttagaatg tattctgtat gcctctcttt tgctctgata
ctcttgcttc 1980ctgctattct gattgtgcag actgtgtaat tagtggaaaa
caatccttgg tctgactgtg 2040actttggaca actcagtaac cctggcttgg
accactctca ggagtccatc cttgagagag 2100tgggtgtagt taccatttat
acagtaatca ttgcatttta aaatctgctc ttgaaaggaa 2160gaataagagt
gcaccagaat aagagcgcac cagaataaga gcgcaccagc taacaatgtg
2220atacggccat atgtcactta aggatagaga tatgttctga gaaatgtgtc
attaggcgat 2280tttgtcatta aacatcatag catgtacttc cacaaaccta
gatggtatag cctactacac 2340acctaggcta tttggtatag cctgttgatc
ctggggtaca aatctgtaca acatgttact 2400gtattgaata cagtaggcaa
ttgtaacaca atggtaagta tctaaacata gaaaagggac 2460agtaaaaata
tggttttata atcttctggg accaccattg tatatgcggt acatcgttga
2520ccaaaacatc gttatccagc atatgactgt atttggttat gaaagccaac
tgttacttga 2580ttctgctttt agttcttaag aggatcaggt ttttaaatac
tcatttacaa gttttctatc 2640ctccttcagt gttaaagtag aaagtaaaaa
gagtatctta tacatgcatg aaattaaagc 2700atataccaaa tgca
271430553PRTPan troglodytes 30Met Ala Asp Pro Glu Val Val Val Ser
Ser Cys Ser Ser His Glu Glu1 5 10 15Glu Asn Arg Cys Asn Phe Asn Gln
Gln Thr Ser Pro Ser Glu Glu Leu 20 25 30Leu Leu Glu Asp Gln Met Arg
Arg Lys Leu Lys Phe Phe Phe Met Asn 35 40 45Pro Cys Glu Lys Phe Trp
Ala Arg Gly Arg Lys Pro Trp Lys Leu Ala 50 55 60Ile Gln Ile Leu Lys
Ile Ala Met Val Thr Ile Gln Leu Val Leu Phe65 70 75 80Gly Leu Ser
Asn Gln Met Val Val Ala Phe Lys Glu Glu Asn Thr Ile 85 90 95Ala Phe
Lys His Leu Phe Leu Lys Gly Tyr Met Asp Arg Met Asp Asp 100 105
110Thr Tyr Ala Val Tyr Thr Gln Ser Asp Val Tyr Asp Gln Leu Ile Phe
115 120 125Ala Val Asn Gln Tyr Leu Gln Leu Tyr Asn Val Ser Val Gly
Asn His 130 135 140Ala Tyr Glu Asn Lys Gly Thr Lys Gln Ser Ala Met
Ala Ile Cys Gln145 150 155 160His Phe Tyr Lys Arg Gly Asn Ile Tyr
Pro Gly Asn Asp Thr Phe Asp 165 170 175Ile Asp Pro Glu Ile Glu Thr
Glu Cys Phe Phe Val Glu Pro Asp Glu 180 185 190Pro Phe His Ile Gly
Thr Pro Ala Glu Asn Lys Leu Asn Leu Thr Leu 195 200 205Asp Phe His
Arg Leu Leu Thr Val Glu Leu Gln Phe Lys Leu Lys Ala 210 215 220Ile
Asn Leu Gln Thr Val Arg His Gln Glu Leu Pro Asp Cys Tyr Asp225 230
235 240Phe Thr Leu Thr Ile Thr Phe Asp Asn Lys Ala His Ser Gly Arg
Ile 245 250 255Lys Ile Ser Leu Asp Asn Asp Ile Ser Ile Arg Glu Cys
Lys Asp Trp 260 265 270His Val Ser Gly Ser Ile Gln Lys Asn Thr His
Tyr Met Met Ile Phe 275 280 285Asp Ala Phe Val Ile Leu Thr Cys Leu
Val Ser Leu Ile Leu Cys Ile 290 295 300Arg Ser Val Ile Arg Gly Leu
Gln Leu Gln Gln Glu Phe Val Asn Phe305 310 315 320Phe Leu Leu His
Tyr Lys Lys Gly Val Ser Val Ser Asp Gln Met Glu 325 330 335Phe Val
Asn Gly Trp Tyr Ile Met Ile Ile Ile Ser Asp Ile Leu Thr 340 345
350Ile Ile Gly Ser Ile Leu Lys Met Glu Ile Gln Ala Lys Ser
Leu Thr 355 360 365Ser Tyr Asp Val Cys Ser Ile Leu Leu Gly Thr Ser
Thr Met Leu Val 370 375 380Trp Leu Gly Val Ile Arg Tyr Leu Gly Phe
Phe Ala Lys Tyr Asn Leu385 390 395 400Leu Ile Leu Thr Leu Gln Ala
Ala Leu Pro Asn Val Ile Arg Phe Cys 405 410 415Cys Cys Ala Ala Met
Ile Tyr Leu Gly Tyr Cys Phe Cys Gly Trp Ile 420 425 430Val Leu Gly
Pro Tyr His Asp Lys Phe Arg Ser Leu Asn Met Val Ser 435 440 445Glu
Cys Leu Phe Ser Leu Ile Asn Gly Asp Asp Met Phe Ala Thr Phe 450 455
460Ala Lys Met Gln Gln Lys Ser Tyr Leu Val Trp Leu Phe Ser Arg
Ile465 470 475 480Tyr Leu Tyr Ser Phe Ile Ser Leu Phe Ile Tyr Met
Ile Leu Ser Leu 485 490 495Phe Ile Ala Leu Ile Thr Asp Thr Tyr Glu
Thr Ile Lys Gln Tyr Gln 500 505 510Gln Asp Gly Phe Pro Glu Thr Glu
Leu Arg Thr Phe Ile Ser Glu Cys 515 520 525Lys Asp Leu Pro Asn Ser
Gly Lys Tyr Arg Leu Glu Asp Asp Pro Pro 530 535 540Val Ser Leu Phe
Cys Cys Cys Lys Lys545 550311896DNASilurana tropicalis 31ttgcaactag
gtctgacagt aggacaatgt ggcaggtcac gtgacagcag tgctgatggt 60agagatgcgc
cagcattcag gtctgagagc agaaagaaaa gctggccaaa acaaaggaca
120ttctctttgc tgcttcgcta gctgagacgc tgctatagta tagcagacat
ggaaaaccca 180gagctaataa agacatgcaa ctctttggat gaacatgatg
gtccctactg ctgcaagcag 240tgccctatga ctgatgagct acttatggaa
gaccagctac gaaggaaact taaattcttt 300ttcatgaacc catgtgagaa
gttccgtgcc cgtggacgaa agccttggaa gctttgtatt 360caaattttaa
aaattgcaat ggtgacaatc caattagttt tatttggact cagtaatgaa
420atggtagtca cctttaaaga ggagaacact gtagctttta agcatctgtt
tttgaaagga 480tataaggatg gacatgatga cacatatgct atctacagtc
aagaagatgt tcatgctcat 540ataaacttta caattaaaag gtacctagag
ctacaaaaca tatctgttgg aaatcatgca 600tatgaaagta atggtaaagg
tcaaactgga atgtcattat gtcaacatta ctataaacaa 660gggagtatct
ttcctggaaa tgaaacattt gaaattgacc cacaaataga tactgaatgt
720ttccatattg atccatcaac tctgtgttct aatgacacac ctgcagaata
ctactggtct 780aatatgacac tagacttcta tagacttgtt tcagttgaaa
ttatgtttaa gcttaaagca 840attaatcttc aaaccattcg tcatcatgaa
cttccagact gctatgactt catggttata 900ataacatttg ataataaggc
acacagtgga aggataaaaa tcagcttaga taatgatgtt 960ggaatccagg
aatgcaaaga ctggcatgtg tctggatcta ttcaaaaaaa tactcattac
1020atgatgattt ttgatgctgc tgttattttg gtctgcttat cttccataac
actctgcatt 1080cgctccgtgg ttaaaggaat tcacctacaa aaagaatatg
taaacttttt ccagcatcgt 1140tttgcaagga ctgtgtcctc agctgatcgc
atggaatttg tcaatggctg gtacattatg 1200ataatcatca gtgatgtttt
gtcaattatt ggctcaatct tgaagatgga gatccaagca 1260aagagcctca
ccagttatga tgtctgcagt attctcttgg gaacatccac cttattagtg
1320tggcttggag ttattcgcta cttgggattt tttaagaaat acaatcttct
gatcctgaca 1380cttagggcag ccttacctaa tgtgatacga ttctgctgtt
gtgctgctat gatctacctg 1440ggctactgct tctgtggctg gattgttctg
gggccttacc atgtaaagtt caggtccctg 1500aacatggttt cagagtgcct
gttctccctt attaatggag acgatatgtt tacaacgttt 1560tcaatcatgc
aggagaagag ctacttggtt tggctgttta gtcgcattta tttgtattcc
1620tttataagtc tcttcatata catggttctg agtctcttca ttgcacttat
tactgacaca 1680tacgatacaa tcaagaatta ccagatcgat ggctttccag
aatcagaact tcacacattt 1740gtatccgagt gcaaagattt gccaacctct
ggtcgatata gggaacaaga cgagacctcc 1800tgtttgtcta tgctgtgttg
taatcggtaa aaaagaatcc cagaagaagc actttatcca 1860tggcctttaa
aaatctgcaa aaaaaaaaaa aaaaaa 189632553PRTSilurana tropicalis 32Met
Glu Asn Pro Glu Leu Ile Lys Thr Cys Asn Ser Leu Asp Glu His1 5 10
15Asp Gly Pro Tyr Cys Cys Lys Gln Cys Pro Met Thr Asp Glu Leu Leu
20 25 30Met Glu Asp Gln Leu Arg Arg Lys Leu Lys Phe Phe Phe Met Asn
Pro 35 40 45Cys Glu Lys Phe Arg Ala Arg Gly Arg Lys Pro Trp Lys Leu
Cys Ile 50 55 60Gln Ile Leu Lys Ile Ala Met Val Thr Ile Gln Leu Val
Leu Phe Gly65 70 75 80Leu Ser Asn Glu Met Val Val Thr Phe Lys Glu
Glu Asn Thr Val Ala 85 90 95Phe Lys His Leu Phe Leu Lys Gly Tyr Lys
Asp Gly His Asp Asp Thr 100 105 110Tyr Ala Ile Tyr Ser Gln Glu Asp
Val His Ala His Ile Asn Phe Thr 115 120 125Ile Lys Arg Tyr Leu Glu
Leu Gln Asn Ile Ser Val Gly Asn His Ala 130 135 140Tyr Glu Ser Asn
Gly Lys Gly Gln Thr Gly Met Ser Leu Cys Gln His145 150 155 160Tyr
Tyr Lys Gln Gly Ser Ile Phe Pro Gly Asn Glu Thr Phe Glu Ile 165 170
175Asp Pro Gln Ile Asp Thr Glu Cys Phe His Ile Asp Pro Ser Thr Leu
180 185 190Cys Ser Asn Asp Thr Pro Ala Glu Tyr Tyr Trp Ser Asn Met
Thr Leu 195 200 205Asp Phe Tyr Arg Leu Val Ser Val Glu Ile Met Phe
Lys Leu Lys Ala 210 215 220Ile Asn Leu Gln Thr Ile Arg His His Glu
Leu Pro Asp Cys Tyr Asp225 230 235 240Phe Met Val Ile Ile Thr Phe
Asp Asn Lys Ala His Ser Gly Arg Ile 245 250 255Lys Ile Ser Leu Asp
Asn Asp Val Gly Ile Gln Glu Cys Lys Asp Trp 260 265 270His Val Ser
Gly Ser Ile Gln Lys Asn Thr His Tyr Met Met Ile Phe 275 280 285Asp
Ala Ala Val Ile Leu Val Cys Leu Ser Ser Ile Thr Leu Cys Ile 290 295
300Arg Ser Val Val Lys Gly Ile His Leu Gln Lys Glu Tyr Val Asn
Phe305 310 315 320Phe Gln His Arg Phe Ala Arg Thr Val Ser Ser Ala
Asp Arg Met Glu 325 330 335Phe Val Asn Gly Trp Tyr Ile Met Ile Ile
Ile Ser Asp Val Leu Ser 340 345 350Ile Ile Gly Ser Ile Leu Lys Met
Glu Ile Gln Ala Lys Ser Leu Thr 355 360 365Ser Tyr Asp Val Cys Ser
Ile Leu Leu Gly Thr Ser Thr Leu Leu Val 370 375 380Trp Leu Gly Val
Ile Arg Tyr Leu Gly Phe Phe Lys Lys Tyr Asn Leu385 390 395 400Leu
Ile Leu Thr Leu Arg Ala Ala Leu Pro Asn Val Ile Arg Phe Cys 405 410
415Cys Cys Ala Ala Met Ile Tyr Leu Gly Tyr Cys Phe Cys Gly Trp Ile
420 425 430Val Leu Gly Pro Tyr His Val Lys Phe Arg Ser Leu Asn Met
Val Ser 435 440 445Glu Cys Leu Phe Ser Leu Ile Asn Gly Asp Asp Met
Phe Thr Thr Phe 450 455 460Ser Ile Met Gln Glu Lys Ser Tyr Leu Val
Trp Leu Phe Ser Arg Ile465 470 475 480Tyr Leu Tyr Ser Phe Ile Ser
Leu Phe Ile Tyr Met Val Leu Ser Leu 485 490 495Phe Ile Ala Leu Ile
Thr Asp Thr Tyr Asp Thr Ile Lys Asn Tyr Gln 500 505 510Ile Asp Gly
Phe Pro Glu Ser Glu Leu His Thr Phe Val Ser Glu Cys 515 520 525Lys
Asp Leu Pro Thr Ser Gly Arg Tyr Arg Glu Gln Asp Glu Thr Ser 530 535
540Cys Leu Ser Met Leu Cys Cys Asn Arg545 550331896DNASilurana
tropicalis 33ttgcaactag gtctgacagt aggacaatgt ggcaggtcac gtgacagcag
tgctgatggt 60agagatgcgc cagcattcag gtctgagagc agaaagaaaa gctggccaaa
acaaaggaca 120ttctctttgc tgcttcgcta gctgagacgc tgctatagta
tagcagacat ggaaaaccca 180gagctaataa agacatgcaa ctctttggat
gaacatgatg gtccctactg ctgcaagcag 240tgccctatga ctgatgagct
acttatggaa gaccagctac gaaggaaact taaattcttt 300ttcatgaacc
catgtgagaa gttccgtgcc cgtggacgaa agccttggaa gctttgtatt
360caaattttaa aaattgcaat ggtgacaatc caattagttt tatttggact
cagtaatgaa 420atggtagtca cctttaaaga ggagaacact gtagctttta
agcatctgtt tttgaaagga 480tataaggatg gacatgatga cacatatgct
atctacagtc aagaagatgt tcatgctcat 540ataaacttta caattaaaag
gtacctagag ctacaaaaca tatctgttgg aaatcatgca 600tatgaaagta
atggtaaagg tcaaactgga atgtcattat gtcaacatta ctataaacaa
660gggagtatct ttcctggaaa tgaaacattt gaaattgacc cacaaataga
tactgaatgt 720ttccatattg atccatcaac tctgtgttct aatgacacac
ctgcagaata ctactggtct 780aatatgacac tagacttcta tagacttgtt
tcagttgaaa ttatgtttaa gcttaaagca 840attaatcttc aaaccattcg
tcatcatgaa cttccagact gctatgactt catggttata 900ataacatttg
ataataaggc acacagtgga aggataaaaa tcagcttaga taatgatgtt
960ggaatccagg aatgcaaaga ctggcatgtg tctggatcta ttcaaaaaaa
tactcattac 1020atgatgattt ttgatgctgc tgttattttg gtctgcttat
cttccataac actctgcatt 1080cgctccgtgg ttaaaggaat tcacctacaa
aaagaatatg taaacttttt ccagcatcgt 1140tttgcaagga ctgtgtcctc
agctgatcgc atggaatttg tcaatggctg gtacattatg 1200ataatcatca
gtgatgtttt gtcaattatt ggctcaatct tgaagatgga gatccaagca
1260aagagcctca ccagttatga tgtctgcagt attctcttgg gaacatccac
cttattagtg 1320tggcttggag ttattcgcta cttgggattt tttaagaaat
acaatcttct gatcctgaca 1380cttagggcag ccttacctaa tgtgatacga
ttctgctgtt gtgctgctat gatctacctg 1440ggctactgct tctgtggctg
gattgttctg gggccttacc atgtaaagtt caggtccctg 1500aacatggttt
cagagtgcct gttctccctt attaatggag acgatatgtt tacaacgttt
1560tcaatcatgc aggagaagag ctacttggtt tggctgttta gtcgcattta
tttgtattcc 1620tttataagtc tcttcatata catggttctg agtctcttca
ttgcacttat tactgacaca 1680tacgatacaa tcaagaatta ccagatcgat
ggctttccag aatcagaact tcacacattt 1740gtatccgagt gcaaagattt
gccaacctct ggtcgatata gggaacaaga cgagacctcc 1800tgtttgtcta
tgctgtgttg taatcggtaa aaaagaatcc cagaagaagc actttatcca
1860tggcctttaa aaatctgcaa aaaaaaaaaa aaaaaa 189634553PRTSilurana
tropicalis 34Met Glu Asn Pro Glu Leu Ile Lys Thr Cys Asn Ser Leu
Asp Glu His1 5 10 15Asp Gly Pro Tyr Cys Cys Lys Gln Cys Pro Met Thr
Asp Glu Leu Leu 20 25 30Met Glu Asp Gln Leu Arg Arg Lys Leu Lys Phe
Phe Phe Met Asn Pro 35 40 45Cys Glu Lys Phe Arg Ala Arg Gly Arg Lys
Pro Trp Lys Leu Cys Ile 50 55 60Gln Ile Leu Lys Ile Ala Met Val Thr
Ile Gln Leu Val Leu Phe Gly65 70 75 80Leu Ser Asn Glu Met Val Val
Thr Phe Lys Glu Glu Asn Thr Val Ala 85 90 95Phe Lys His Leu Phe Leu
Lys Gly Tyr Lys Asp Gly His Asp Asp Thr 100 105 110Tyr Ala Ile Tyr
Ser Gln Glu Asp Val His Ala His Ile Asn Phe Thr 115 120 125Ile Lys
Arg Tyr Leu Glu Leu Gln Asn Ile Ser Val Gly Asn His Ala 130 135
140Tyr Glu Ser Asn Gly Lys Gly Gln Thr Gly Met Ser Leu Cys Gln
His145 150 155 160Tyr Tyr Lys Gln Gly Ser Ile Phe Pro Gly Asn Glu
Thr Phe Glu Ile 165 170 175Asp Pro Gln Ile Asp Thr Glu Cys Phe His
Ile Asp Pro Ser Thr Leu 180 185 190Cys Ser Asn Asp Thr Pro Ala Glu
Tyr Tyr Trp Ser Asn Met Thr Leu 195 200 205Asp Phe Tyr Arg Leu Val
Ser Val Glu Ile Met Phe Lys Leu Lys Ala 210 215 220Ile Asn Leu Gln
Thr Ile Arg His His Glu Leu Pro Asp Cys Tyr Asp225 230 235 240Phe
Met Val Ile Ile Thr Phe Asp Asn Lys Ala His Ser Gly Arg Ile 245 250
255Lys Ile Ser Leu Asp Asn Asp Val Gly Ile Gln Glu Cys Lys Asp Trp
260 265 270His Val Ser Gly Ser Ile Gln Lys Asn Thr His Tyr Met Met
Ile Phe 275 280 285Asp Ala Ala Val Ile Leu Val Cys Leu Ser Ser Ile
Thr Leu Cys Ile 290 295 300Arg Ser Val Val Lys Gly Ile His Leu Gln
Lys Glu Tyr Val Asn Phe305 310 315 320Phe Gln His Arg Phe Ala Arg
Thr Val Ser Ser Ala Asp Arg Met Glu 325 330 335Phe Val Asn Gly Trp
Tyr Ile Met Ile Ile Ile Ser Asp Val Leu Ser 340 345 350Ile Ile Gly
Ser Ile Leu Lys Met Glu Ile Gln Ala Lys Ser Leu Thr 355 360 365Ser
Tyr Asp Val Cys Ser Ile Leu Leu Gly Thr Ser Thr Leu Leu Val 370 375
380Trp Leu Gly Val Ile Arg Tyr Leu Gly Phe Phe Lys Lys Tyr Asn
Leu385 390 395 400Leu Ile Leu Thr Leu Arg Ala Ala Leu Pro Asn Val
Ile Arg Phe Cys 405 410 415Cys Cys Ala Ala Met Ile Tyr Leu Gly Tyr
Cys Phe Cys Gly Trp Ile 420 425 430Val Leu Gly Pro Tyr His Val Lys
Phe Arg Ser Leu Asn Met Val Ser 435 440 445Glu Cys Leu Phe Ser Leu
Ile Asn Gly Asp Asp Met Phe Thr Thr Phe 450 455 460Ser Ile Met Gln
Glu Lys Ser Tyr Leu Val Trp Leu Phe Ser Arg Ile465 470 475 480Tyr
Leu Tyr Ser Phe Ile Ser Leu Phe Ile Tyr Met Val Leu Ser Leu 485 490
495Phe Ile Ala Leu Ile Thr Asp Thr Tyr Asp Thr Ile Lys Asn Tyr Gln
500 505 510Ile Asp Gly Phe Pro Glu Ser Glu Leu His Thr Phe Val Ser
Glu Cys 515 520 525Lys Asp Leu Pro Thr Ser Gly Arg Tyr Arg Glu Gln
Asp Glu Thr Ser 530 535 540Cys Leu Ser Met Leu Cys Cys Asn Arg545
550356923DNAHomo sapiens 35cgcgctgcct gagctgagcc gccgtaggtg
aggggcccgc gtccccgccc gccctgggcg 60ccgcgcctgg cactgatcct gccggtcgcc
cactgtcgcc gccgccgccg cccgcgggca 120ccatgacagc tctgagcgct
ggggttacag actgtggttt tgtgcttgct caccaaagct 180aacctcagca
tgctcaaaag gaagcagagt tccagggtgg aagcccagcc agtcactgac
240tttggtcctg atgagtctct gtcggataat gctgacatcc tctggattaa
caaaccatgg 300gttcactctt tgctgcgcat ctgtgccatc atcagcgtca
tttctgtttg tatgaatacg 360ccaatgacct tcgagcacta tcctccactt
cagtatgtga ccttcacttt ggatacatta 420ttgatgtttc tctacacggc
agagatgata gcaaaaatgc acatccgggg cattgtcaag 480ggggatagtt
cctatgtgaa agatcgctgg tgtgtttttg atggatttat ggtcttttgc
540ctttgggttt ctttggtgct acaggtgttt gaaattgctg atatagttga
tcagatgtca 600ccttggggca tgttgcggat tccacggcca ctgattatga
tccgagcatt ccggatttat 660ttccgatttg aactgccaag gaccagaatt
acaaatattt taaagcgatc gggagaacaa 720atatggagtg tttccatttt
tctacttttc tttctacttc tttatggaat tttaggagtt 780cagatgtttg
gaacatttac ttatcactgt gttgtaaatg acacaaagcc agggaatgta
840acctggaata gtttagctat tccagacaca cactgctcac cagagctaga
agaaggctac 900cagtgcccac ctggatttaa atgcatggac cttgaagatc
tgggacttag caggcaagag 960ctgggctaca gtggctttaa tgagatagga
actagtatat tcaccgtcta tgaggccgcc 1020tcacaggaag gctgggtgtt
cctcatgtac agagcaattg acagctttcc ccgttggcgt 1080tcctacttct
atttcatcac tctcattttc ttcctcgcct ggcttgtgaa gaacgtgttt
1140attgctgtta tcattgaaac atttgcagaa atcagagtac agtttcaaca
aatgtgggga 1200tcgagaagca gcactacctc aacagccacc acccagatgt
ttcatgaaga tgctgctgga 1260ggttggcagc tggtagctgt ggatgtcaac
aagccccagg gacgcgcccc agcctgcctc 1320cagaaaatga tgcggtcatc
cgttttccac atgttcatcc tgagcatggt gaccgtggac 1380gtgatcgtgg
cggctagcaa ctactacaaa ggagaaaact tcaggaggca gtacgacgag
1440ttctacctgg cggaggtggc ttttacagta ctttttgatt tggaagcact
tctgaagata 1500tggtgtttgg gatttactgg atatattagc tcatctctcc
acaaattcga actactactc 1560gtaattggaa ctactcttca tgtataccca
gatctttatc attcacaatt cacgtacttt 1620caggttctcc gagtagttcg
gctgattaag atttcacctg cattagaaga ctttgtgtac 1680aagatatttg
gtcctggaaa aaagcttggg agtttggttg tatttactgc cagcctcttg
1740attgttatgt cagcaattag tttgcagatg ttctgctttg tcgaagaact
ggacagattt 1800actacgtttc cgagggcatt tatgtccatg ttccagatcc
tcacccagga aggatgggtg 1860gacgtaatgg accaaactct aaatgctgtg
ggacatatgt gggcacccgt ggttgccatc 1920tatttcattc tctatcatct
ttttgccact ctgatcctcc tgagtttgtt tgttgctgtt 1980attttggaca
acttagaact tgatgaagac ctaaagaagc ttaaacaatt aaagcaaagt
2040gaagcaaatg cggacaccaa agaaaagctc cctttacgcc tgcgaatctt
tgaaaaattt 2100ccaaacagac ctcaaatggt gaaaatctca aagcttcctt
cagattttac agttcctaaa 2160atcagggaga gttttatgaa gcagtttatt
gaccgccagc aacaggacac atgttgcctc 2220ctgagaagcc tcccgaccac
ctcttcctcc tcctgcgacc actccaaacg ctcagcaatt 2280gaggacaaca
aatacatcga ccaaaaactt cgcaagtctg ttttcagcat cagggcaagg
2340aaccttctgg aaaaggagac cgcagtcact aaaatcttaa gagcttgcac
ccgacagcgc 2400atgctgagcg gatcatttga ggggcagccc gcaaaggaga
ggtcaatcct cagcgtgcag 2460catcatatcc gccaagagcg caggtcacta
agacatggat caaacagcca gaggatcagc 2520aggggaaaat ctcttgaaac
tttgactcaa gatcattcca atacagtgag atatagaaat 2580gcacaaagag
aagacagtga aataaagatg attcaggaaa aaaaggagca agcagagatg
2640aaaaggaaag tgcaagaaga ggaactcaga gagaaccacc catacttcga
taagccactg 2700ttcattgtcg ggcgagaaca caggttcaga aacttttgcc
gggtggtggt ccgagcacgc 2760ttcaacgcat ctaaaacaga ccctgtcaca
ggagctgtga aaaatacaaa gtaccatcaa 2820ctttatgatt tgctgggatt
ggtcacttac ctggactggg tcatgatcat cgtaaccatc 2880tgctcttgca
tttccatgat gtttgagtcc ccgtttcgaa gagtcatgca tgcacctact
2940ttgcagattg ctgagtatgt gtttgtgata ttcatgagca ttgagcttaa
tctgaagatt 3000atggcagatg gcttattttt cactccaact gctgtcatca
gggacttcgg tggagtaatg 3060gacatattta tatatcttgt gagcttgata
tttctttgtt ggatgcctca aaatgtacct 3120gctgaatcgg gagctcagct
tctaatggtc cttcggtgcc tgagacctct gcgcatattc 3180aaactggtgc
cccagatgag gaaagttgtt cgagaacttt
tcagcggctt caaggaaatt 3240tttttggtct ccattctttt gctgacatta
atgctcgttt ttgcaagctt tggagttcag 3300ctttttgctg gaaaactggc
caagtgcaat gatcccaaca ttattagaag ggaagattgc 3360aatggcatat
tcagaattaa tgtcagtgtg tcaaagaact taaatttaaa attgaggcct
3420ggagagaaaa aacctggatt ttgggtgccc cgtgtttggg cgaatcctcg
gaactttaat 3480ttcgacaatg tgggaaacgc tatgctggcg ttgtttgaag
ttctctcctt gaaaggctgg 3540gtggaagtga gagatgttat tattcatcgt
gtggggccga tccatggaat ctatattcat 3600gtttttgtat tcctgggttg
catgattgga ctgacccttt ttgttggagt agttattgct 3660aatttcaatg
aaaacaaggg gacggctttg ctgaccgtcg atcagagaag atgggaagac
3720ctgaagagcc gactgaagat cgcacagcct cttcatcttc cgcctcgccc
ggataatgat 3780ggttttagag ctaaaatgta tgacataacc cagcatccat
tttttaagag gacaatcgca 3840ttactcgtcc tggcccagtc ggtgttgctc
tctgtcaagt gggacgtcga ggacccggtg 3900accgtacctt tggcaacaat
gtcagttgtt ttcaccttca tctttgttct ggaggttacc 3960atgaagatca
tagcaatgtc gcctgctggc ttctggcaaa gcagaagaaa ccgatacgat
4020ctcctggtga cgtcgcttgg cgttgtatgg gtggtgcttc actttgccct
cctgaatgca 4080tatacttaca tgatgggcgc ttgtgtgatt gtatttaggt
ttttctccat ctgtggaaaa 4140catgtaacgc taaagatgct cctcttgaca
gtggtcgtca gcatgtacaa gagcttcttt 4200atcatagtag gcatgtttct
cttgctgctg tgttacgctt ttgctggagt tgttttattt 4260ggtactgtga
aatatgggga gaatattaac aggcatgcaa atttttcttc ggctggaaaa
4320gctattaccg tactgttccg aattgtcaca ggtgaagact ggaacaagat
tatgcatgac 4380tgtatggttc agcctccgtt ttgtactcca gatgaattta
catactgggc aacagactgt 4440ggaaattatg ctggggcact tatgtatttc
tgttcatttt atgtcatcat tgcctacatc 4500atgctaaatc tgcttgtagc
cataattgtg gagaatttct ccttgtttta ttccactgag 4560gaggaccagc
ttttaagtta caatgatctt cgccactttc aaataatatg gaacatggtg
4620gatgataaaa gagagggggt gatccccacg ttccgcgtca agttcctgct
gcggctactg 4680cgtgggaggc tggaggtgga cctggacaag gacaagctcc
tgtttaagca catgtgctac 4740gaaatggaga ggctccacaa tggcggcgac
gtcaccttcc atgatgtcct gagcatgctt 4800tcataccggt ccgtggacat
ccggaagagc ttgcagctgg aggaactcct ggcgagggag 4860cagctggagt
acaccataga ggaggaggtg gccaagcaga ccatccgcat gtggctcaag
4920aagtgcctga agcgcatcag agctaaacag cagcagtcgt gcagtatcat
ccacagcctg 4980agagagagtc agcagcaaga gctgagccgg tttctgaacc
cgcccagcat cgagaccacc 5040cagcccagtg aggacacgaa tgccaacagt
caggacaaca gcatgcaacc tgagacaagc 5100agccagcagc agctcctgag
ccccacgctg tcggatcgag gaggaagtcg gcaagatgca 5160gccgacgcag
ggaaacccca gaggaaattt gggcagtggc gtctgccctc agccccaaaa
5220ccaataagcc attcagtgtc ctcagtcaac ttacggtttg gaggaaggac
aaccatgaaa 5280tctgtcgtgt gcaaaatgaa ccccatgact gacgcggctt
cctgcggttc tgaagttaag 5340aagtggtgga cccggcagct gactgtggag
agcgacgaaa gtggggatga ccttctggat 5400atttaggtgg atgtcaatgt
agatgaattt ctagtggtgg aaaccgtttt ctaataatgt 5460ccttgattgt
ccagtgagca atctgtaatt gatctataac tgaattccag cttgtcacaa
5520gatgtttata aattgatttt catcctgcca cagaaaggca taagctgcat
gtatgatggg 5580ttactatcaa tcattgctca aaaaaatttt tgtataatga
cagtactgat aatattagaa 5640atgataccgc aagcaaatgt atatcactta
aaaatgtcat atattctgtc tgcgtaaact 5700aaggtatata ttcatatgtg
ctctaatgca gtattatcac cgccccgcaa aagagtgcta 5760agcccaaagt
ggctgatatt tagggtacag gggttatagc tttagttcac atctttccca
5820tttccactag aaatatttct cttgagagaa tttattattt atgattgatc
tgaaaaggtc 5880agcactgaac ttatgctaaa atgatagtag ttttacaaac
tacagattct gaattttaaa 5940aagtatcttc tttttctcgt gttatatttt
taaatataca caagacattt ggtgaccaga 6000acaagttgat ttctgtcctc
agttatgtta atgaaactgt tgcctccttc taagaaaatt 6060gtgtgtgcaa
gcaccaggca aagaaatgga ctcaggatgc ttagcggttt aaaacaaacc
6120tgtagataaa tcacttgagt gacatagttg cgcaaagatg ttaagtttct
taagaaacct 6180tttaataact gagtttagca aaaagaataa aactatatag
ctcaatttat ttaaaaaaat 6240ctttgcatgt gtgatgttat cattggcttc
atttcttacc caaggtatgt ctgttttgcc 6300ataaatcagc agagtcattt
cattctgggt gatcctaaca caccattgct acgttagatt 6360tgaaatgaca
tctctgttaa aagaatcttc tatggaaata atggtgccct gcaaaatctt
6420cctttgaact cacaggttag ggatcacaca acttacttaa tcgttttttg
tttttgtttt 6480ttttccttat atgtcaatgg cccatgtcct ccgggaaaat
tagaaaagca aaatgattac 6540aaagtgctgt tagatttctt gtgctgggcc
agccaagtag aagtggactt gacttggacc 6600tttaactatt ttattacaga
ttggacattt gctgttcaga tgttttttaa cagagggatt 6660atctcagaat
cctgtgacct ccaggttgtt ttataatcta tttttctcta tttaacattc
6720ctcagataga taggcaaata ggacattcct tctgtgtcac agaagtatcg
tggtagtggc 6780agtctacagt ttatatgatt cattgtaact atgagataaa
gaacaaccag tcatgtggcc 6840aaaaggatta gatttgattt gatgttcact
tggagtttac tttttgtaca tacaagataa 6900aataaatatt ggatttgtaa aat
6923361738PRTHomo sapiens 36Met Leu Lys Arg Lys Gln Ser Ser Arg Val
Glu Ala Gln Pro Val Thr1 5 10 15Asp Phe Gly Pro Asp Glu Ser Leu Ser
Asp Asn Ala Asp Ile Leu Trp 20 25 30Ile Asn Lys Pro Trp Val His Ser
Leu Leu Arg Ile Cys Ala Ile Ile 35 40 45Ser Val Ile Ser Val Cys Met
Asn Thr Pro Met Thr Phe Glu His Tyr 50 55 60Pro Pro Leu Gln Tyr Val
Thr Phe Thr Leu Asp Thr Leu Leu Met Phe65 70 75 80Leu Tyr Thr Ala
Glu Met Ile Ala Lys Met His Ile Arg Gly Ile Val 85 90 95Lys Gly Asp
Ser Ser Tyr Val Lys Asp Arg Trp Cys Val Phe Asp Gly 100 105 110Phe
Met Val Phe Cys Leu Trp Val Ser Leu Val Leu Gln Val Phe Glu 115 120
125Ile Ala Asp Ile Val Asp Gln Met Ser Pro Trp Gly Met Leu Arg Ile
130 135 140Pro Arg Pro Leu Ile Met Ile Arg Ala Phe Arg Ile Tyr Phe
Arg Phe145 150 155 160Glu Leu Pro Arg Thr Arg Ile Thr Asn Ile Leu
Lys Arg Ser Gly Glu 165 170 175Gln Ile Trp Ser Val Ser Ile Phe Leu
Leu Phe Phe Leu Leu Leu Tyr 180 185 190Gly Ile Leu Gly Val Gln Met
Phe Gly Thr Phe Thr Tyr His Cys Val 195 200 205Val Asn Asp Thr Lys
Pro Gly Asn Val Thr Trp Asn Ser Leu Ala Ile 210 215 220Pro Asp Thr
His Cys Ser Pro Glu Leu Glu Glu Gly Tyr Gln Cys Pro225 230 235
240Pro Gly Phe Lys Cys Met Asp Leu Glu Asp Leu Gly Leu Ser Arg Gln
245 250 255Glu Leu Gly Tyr Ser Gly Phe Asn Glu Ile Gly Thr Ser Ile
Phe Thr 260 265 270Val Tyr Glu Ala Ala Ser Gln Glu Gly Trp Val Phe
Leu Met Tyr Arg 275 280 285Ala Ile Asp Ser Phe Pro Arg Trp Arg Ser
Tyr Phe Tyr Phe Ile Thr 290 295 300Leu Ile Phe Phe Leu Ala Trp Leu
Val Lys Asn Val Phe Ile Ala Val305 310 315 320Ile Ile Glu Thr Phe
Ala Glu Ile Arg Val Gln Phe Gln Gln Met Trp 325 330 335Gly Ser Arg
Ser Ser Thr Thr Ser Thr Ala Thr Thr Gln Met Phe His 340 345 350Glu
Asp Ala Ala Gly Gly Trp Gln Leu Val Ala Val Asp Val Asn Lys 355 360
365Pro Gln Gly Arg Ala Pro Ala Cys Leu Gln Lys Met Met Arg Ser Ser
370 375 380Val Phe His Met Phe Ile Leu Ser Met Val Thr Val Asp Val
Ile Val385 390 395 400Ala Ala Ser Asn Tyr Tyr Lys Gly Glu Asn Phe
Arg Arg Gln Tyr Asp 405 410 415Glu Phe Tyr Leu Ala Glu Val Ala Phe
Thr Val Leu Phe Asp Leu Glu 420 425 430Ala Leu Leu Lys Ile Trp Cys
Leu Gly Phe Thr Gly Tyr Ile Ser Ser 435 440 445Ser Leu His Lys Phe
Glu Leu Leu Leu Val Ile Gly Thr Thr Leu His 450 455 460Val Tyr Pro
Asp Leu Tyr His Ser Gln Phe Thr Tyr Phe Gln Val Leu465 470 475
480Arg Val Val Arg Leu Ile Lys Ile Ser Pro Ala Leu Glu Asp Phe Val
485 490 495Tyr Lys Ile Phe Gly Pro Gly Lys Lys Leu Gly Ser Leu Val
Val Phe 500 505 510Thr Ala Ser Leu Leu Ile Val Met Ser Ala Ile Ser
Leu Gln Met Phe 515 520 525Cys Phe Val Glu Glu Leu Asp Arg Phe Thr
Thr Phe Pro Arg Ala Phe 530 535 540Met Ser Met Phe Gln Ile Leu Thr
Gln Glu Gly Trp Val Asp Val Met545 550 555 560Asp Gln Thr Leu Asn
Ala Val Gly His Met Trp Ala Pro Val Val Ala 565 570 575Ile Tyr Phe
Ile Leu Tyr His Leu Phe Ala Thr Leu Ile Leu Leu Ser 580 585 590Leu
Phe Val Ala Val Ile Leu Asp Asn Leu Glu Leu Asp Glu Asp Leu 595 600
605Lys Lys Leu Lys Gln Leu Lys Gln Ser Glu Ala Asn Ala Asp Thr Lys
610 615 620Glu Lys Leu Pro Leu Arg Leu Arg Ile Phe Glu Lys Phe Pro
Asn Arg625 630 635 640Pro Gln Met Val Lys Ile Ser Lys Leu Pro Ser
Asp Phe Thr Val Pro 645 650 655Lys Ile Arg Glu Ser Phe Met Lys Gln
Phe Ile Asp Arg Gln Gln Gln 660 665 670Asp Thr Cys Cys Leu Leu Arg
Ser Leu Pro Thr Thr Ser Ser Ser Ser 675 680 685Cys Asp His Ser Lys
Arg Ser Ala Ile Glu Asp Asn Lys Tyr Ile Asp 690 695 700Gln Lys Leu
Arg Lys Ser Val Phe Ser Ile Arg Ala Arg Asn Leu Leu705 710 715
720Glu Lys Glu Thr Ala Val Thr Lys Ile Leu Arg Ala Cys Thr Arg Gln
725 730 735Arg Met Leu Ser Gly Ser Phe Glu Gly Gln Pro Ala Lys Glu
Arg Ser 740 745 750Ile Leu Ser Val Gln His His Ile Arg Gln Glu Arg
Arg Ser Leu Arg 755 760 765His Gly Ser Asn Ser Gln Arg Ile Ser Arg
Gly Lys Ser Leu Glu Thr 770 775 780Leu Thr Gln Asp His Ser Asn Thr
Val Arg Tyr Arg Asn Ala Gln Arg785 790 795 800Glu Asp Ser Glu Ile
Lys Met Ile Gln Glu Lys Lys Glu Gln Ala Glu 805 810 815Met Lys Arg
Lys Val Gln Glu Glu Glu Leu Arg Glu Asn His Pro Tyr 820 825 830Phe
Asp Lys Pro Leu Phe Ile Val Gly Arg Glu His Arg Phe Arg Asn 835 840
845Phe Cys Arg Val Val Val Arg Ala Arg Phe Asn Ala Ser Lys Thr Asp
850 855 860Pro Val Thr Gly Ala Val Lys Asn Thr Lys Tyr His Gln Leu
Tyr Asp865 870 875 880Leu Leu Gly Leu Val Thr Tyr Leu Asp Trp Val
Met Ile Ile Val Thr 885 890 895Ile Cys Ser Cys Ile Ser Met Met Phe
Glu Ser Pro Phe Arg Arg Val 900 905 910Met His Ala Pro Thr Leu Gln
Ile Ala Glu Tyr Val Phe Val Ile Phe 915 920 925Met Ser Ile Glu Leu
Asn Leu Lys Ile Met Ala Asp Gly Leu Phe Phe 930 935 940Thr Pro Thr
Ala Val Ile Arg Asp Phe Gly Gly Val Met Asp Ile Phe 945 950 955
960Ile Tyr Leu Val Ser Leu Ile Phe Leu Cys Trp Met Pro Gln Asn Val
965 970 975Pro Ala Glu Ser Gly Ala Gln Leu Leu Met Val Leu Arg Cys
Leu Arg 980 985 990Pro Leu Arg Ile Phe Lys Leu Val Pro Gln Met Arg
Lys Val Val Arg 995 1000 1005Glu Leu Phe Ser Gly Phe Lys Glu Ile
Phe Leu Val Ser Ile Leu1010 1015 1020Leu Leu Thr Leu Met Leu Val
Phe Ala Ser Phe Gly Val Gln Leu1025 1030 1035Phe Ala Gly Lys Leu
Ala Lys Cys Asn Asp Pro Asn Ile Ile Arg1040 1045 1050Arg Glu Asp
Cys Asn Gly Ile Phe Arg Ile Asn Val Ser Val Ser1055 1060 1065Lys
Asn Leu Asn Leu Lys Leu Arg Pro Gly Glu Lys Lys Pro Gly1070 1075
1080Phe Trp Val Pro Arg Val Trp Ala Asn Pro Arg Asn Phe Asn Phe1085
1090 1095Asp Asn Val Gly Asn Ala Met Leu Ala Leu Phe Glu Val Leu
Ser1100 1105 1110Leu Lys Gly Trp Val Glu Val Arg Asp Val Ile Ile
His Arg Val1115 1120 1125Gly Pro Ile His Gly Ile Tyr Ile His Val
Phe Val Phe Leu Gly1130 1135 1140Cys Met Ile Gly Leu Thr Leu Phe
Val Gly Val Val Ile Ala Asn1145 1150 1155Phe Asn Glu Asn Lys Gly
Thr Ala Leu Leu Thr Val Asp Gln Arg1160 1165 1170Arg Trp Glu Asp
Leu Lys Ser Arg Leu Lys Ile Ala Gln Pro Leu1175 1180 1185His Leu
Pro Pro Arg Pro Asp Asn Asp Gly Phe Arg Ala Lys Met1190 1195
1200Tyr Asp Ile Thr Gln His Pro Phe Phe Lys Arg Thr Ile Ala Leu1205
1210 1215Leu Val Leu Ala Gln Ser Val Leu Leu Ser Val Lys Trp Asp
Val1220 1225 1230Glu Asp Pro Val Thr Val Pro Leu Ala Thr Met Ser
Val Val Phe1235 1240 1245Thr Phe Ile Phe Val Leu Glu Val Thr Met
Lys Ile Ile Ala Met1250 1255 1260Ser Pro Ala Gly Phe Trp Gln Ser
Arg Arg Asn Arg Tyr Asp Leu1265 1270 1275Leu Val Thr Ser Leu Gly
Val Val Trp Val Val Leu His Phe Ala1280 1285 1290Leu Leu Asn Ala
Tyr Thr Tyr Met Met Gly Ala Cys Val Ile Val1295 1300 1305Phe Arg
Phe Phe Ser Ile Cys Gly Lys His Val Thr Leu Lys Met1310 1315
1320Leu Leu Leu Thr Val Val Val Ser Met Tyr Lys Ser Phe Phe Ile1325
1330 1335Ile Val Gly Met Phe Leu Leu Leu Leu Cys Tyr Ala Phe Ala
Gly1340 1345 1350Val Val Leu Phe Gly Thr Val Lys Tyr Gly Glu Asn
Ile Asn Arg1355 1360 1365His Ala Asn Phe Ser Ser Ala Gly Lys Ala
Ile Thr Val Leu Phe1370 1375 1380Arg Ile Val Thr Gly Glu Asp Trp
Asn Lys Ile Met His Asp Cys1385 1390 1395Met Val Gln Pro Pro Phe
Cys Thr Pro Asp Glu Phe Thr Tyr Trp1400 1405 1410Ala Thr Asp Cys
Gly Asn Tyr Ala Gly Ala Leu Met Tyr Phe Cys1415 1420 1425Ser Phe
Tyr Val Ile Ile Ala Tyr Ile Met Leu Asn Leu Leu Val1430 1435
1440Ala Ile Ile Val Glu Asn Phe Ser Leu Phe Tyr Ser Thr Glu Glu1445
1450 1455Asp Gln Leu Leu Ser Tyr Asn Asp Leu Arg His Phe Gln Ile
Ile1460 1465 1470Trp Asn Met Val Asp Asp Lys Arg Glu Gly Val Ile
Pro Thr Phe1475 1480 1485Arg Val Lys Phe Leu Leu Arg Leu Leu Arg
Gly Arg Leu Glu Val1490 1495 1500Asp Leu Asp Lys Asp Lys Leu Leu
Phe Lys His Met Cys Tyr Glu1505 1510 1515Met Glu Arg Leu His Asn
Gly Gly Asp Val Thr Phe His Asp Val1520 1525 1530Leu Ser Met Leu
Ser Tyr Arg Ser Val Asp Ile Arg Lys Ser Leu1535 1540 1545Gln Leu
Glu Glu Leu Leu Ala Arg Glu Gln Leu Glu Tyr Thr Ile1550 1555
1560Glu Glu Glu Val Ala Lys Gln Thr Ile Arg Met Trp Leu Lys Lys1565
1570 1575Cys Leu Lys Arg Ile Arg Ala Lys Gln Gln Gln Ser Cys Ser
Ile1580 1585 1590Ile His Ser Leu Arg Glu Ser Gln Gln Gln Glu Leu
Ser Arg Phe1595 1600 1605Leu Asn Pro Pro Ser Ile Glu Thr Thr Gln
Pro Ser Glu Asp Thr1610 1615 1620Asn Ala Asn Ser Gln Asp Asn Ser
Met Gln Pro Glu Thr Ser Ser1625 1630 1635Gln Gln Gln Leu Leu Ser
Pro Thr Leu Ser Asp Arg Gly Gly Ser1640 1645 1650Arg Gln Asp Ala
Ala Asp Ala Gly Lys Pro Gln Arg Lys Phe Gly1655 1660 1665Gln Trp
Arg Leu Pro Ser Ala Pro Lys Pro Ile Ser His Ser Val1670 1675
1680Ser Ser Val Asn Leu Arg Phe Gly Gly Arg Thr Thr Met Lys Ser1685
1690 1695Val Val Cys Lys Met Asn Pro Met Thr Asp Ala Ala Ser Cys
Gly1700 1705 1710Ser Glu Val Lys Lys Trp Trp Thr Arg Gln Leu Thr
Val Glu Ser1715 1720 1725Asp Glu Ser Gly Asp Asp Leu Leu Asp
Ile1730 1735371566DNAHomo sapiens 37agccgcgagc ggcggccgcg
gggccgagga gcctgggccg ggccgggcgg ggactactcc 60ggagtcagga ggcagcagcg
gcggaggacg aggatctctg gcagtcagcg ccgctcggac 120gccgccggca
ccatgggctg ctgcaccgga cgctgctcgc tcatctgcct ctgcgcgctg
180cagttggtct cagcattaga gaggcagatc tttgacttcc ttggtttcca
gtgggcgcct 240attcttggaa attttctaca cataatagtt gtcatattgg
gtttgtttgg gaccattcag 300tacagacctc gatacataat ggtgtataca
gtgtggactg ccctctgggt cacctggaat 360gtgttcatta tctgctttta
tttggaagta ggtggactct caaaggacac cgatctaatg 420acattcaata
tctctgtaca tcggtcatgg tggagagaac atgggcctgg ttgtgtcaga
480agagtgctgc ctccctcagc ccatggcatg atggacgatt acacgtacgt
ctctgtcaca 540ggctgcatcg ttgacttcca gtacctggag gtcatccaca
gtgctgtcca aatactactc 600tctttggtgg gttttgtgta tgcctgttat
gtgatcagta tttccatgga agaagaagac 660acatattcat gtgatctgca
agtatgcaaa catcttttta tccagatgct gcaaattatt 720gaataagcaa
gaattagtaa gatattatca ccaaattgtc
acatcagtca agcctcatgt 780gcttcctaag aactgaggtg atgcattatt
ttagagtgtc attctaaacc ccagattcaa 840catcttccta atctttctag
tgcagtctaa tatataaatt ttatgaaaag cataggtttt 900tttttaacca
gcagtgctct ttgagaattt acattgattc ctaaagattg ccattgcttt
960gtataaaatg ttataaatta tcttagcatc ttacctggaa tttccactaa
attcaccaat 1020ttatgatttg tgaaatctga ttttactttt tgaaaatttt
catgtgaatt tcccattttc 1080agtgttgtag cacctctctc ttcctctaag
atcctccaag ctcatcaaaa gccatgatct 1140tattatacca gcagttttat
ttattcaatc tttcaacaag tagttattga acttctataa 1200tgtgccaggc
tctggagctc gccttacacc aaacagacac aatcgatcca ttcgaagtgt
1260cgtaattaca cattgaggga ccaactagac cttttctcat tgtaaacttg
gagcaaaagt 1320aaattcatta aaataaattt acattatagt gccacaaaaa
aatgaacaga accagaaagc 1380attttttaca aaaattaaca gaacagtgtg
atagagggga aaaggatgtg agatcatggt 1440gccctacctt caatagggtg
gccagaaaac acctctctga agaagcagca tttgagctga 1500gacctgaaga
acgaggagtc agtgatgcag agaacctcag gagatgcctt ccaatctgag 1560aaaaga
156638197PRTHomo sapiens 38Met Gly Cys Cys Thr Gly Arg Cys Ser Leu
Ile Cys Leu Cys Ala Leu1 5 10 15Gln Leu Val Ser Ala Leu Glu Arg Gln
Ile Phe Asp Phe Leu Gly Phe 20 25 30Gln Trp Ala Pro Ile Leu Gly Asn
Phe Leu His Ile Ile Val Val Ile 35 40 45Leu Gly Leu Phe Gly Thr Ile
Gln Tyr Arg Pro Arg Tyr Ile Met Val 50 55 60Tyr Thr Val Trp Thr Ala
Leu Trp Val Thr Trp Asn Val Phe Ile Ile65 70 75 80Cys Phe Tyr Leu
Glu Val Gly Gly Leu Ser Lys Asp Thr Asp Leu Met 85 90 95Thr Phe Asn
Ile Ser Val His Arg Ser Trp Trp Arg Glu His Gly Pro 100 105 110Gly
Cys Val Arg Arg Val Leu Pro Pro Ser Ala His Gly Met Met Asp 115 120
125Asp Tyr Thr Tyr Val Ser Val Thr Gly Cys Ile Val Asp Phe Gln Tyr
130 135 140Leu Glu Val Ile His Ser Ala Val Gln Ile Leu Leu Ser Leu
Val Gly145 150 155 160Phe Val Tyr Ala Cys Tyr Val Ile Ser Ile Ser
Met Glu Glu Glu Asp 165 170 175Thr Tyr Ser Cys Asp Leu Gln Val Cys
Lys His Leu Phe Ile Gln Met 180 185 190Leu Gln Ile Ile Glu
195392958DNAHomo sapiens 39tggagtcgct cgctgactcg ccctgcgccc
tcgccgcgga caccggagct gcggccgctc 60cccgctgtcc cccagcttac tccaatcaag
cctctgcccg ccaggaacag gtaacctgtg 120tgtgtccgtt tgctccttct
aagagcatgc ctgatagata cttcggtagc ctctccggat 180ggccccttcg
tcgggtagcc tctcctgatg gggtccttcg cccaccctgc ctcccgcgcc
240ggcgctccgg gtgaatgtca agggtggctg gctgcgaata cctccttcag
ctgctggggt 300tcccgacagt ttgcagtttt taaaagtgca ccctcggaag
ggcttttcag actgggtaaa 360cctgactttt ccaagagatg gcagatcctg
aggtagttgt gagtagctgc agctctcatg 420aagaggaaaa tcgctgcaat
tttaaccagc aaacatctcc atctgaggag cttctattag 480aagaccagat
gaggcgaaaa ctcaaatttt ttttcatgaa tccctgtgag aagttctggg
540ctcgaggtag aaaaccatgg aaacttgcca tacaaattct aaaaattgca
atggtgacta 600tccagctggt cttatttggg ctaagtaacc agatggtggt
agctttcaag gaagagaata 660ctatagcatt caaacacctt ttcctaaaag
gatatatgga ccgaatggat gacacatatg 720cagtgtacac acaaagtgac
gtgtatgatc agttaatctt cgcagtaaac cagtacttgc 780agctatacaa
tgtctccgtt gggaatcatg cttatgagaa caaaggtacc aagcaatctg
840ctatggcaat ctgtcagcac ttatacaagc gaggaaacat ctaccctgga
aatgatacct 900ttgacatcga tccagaaatt gaaactgagt gtttctttgt
ggagccagat gaaccttttc 960acattgggac accagcagaa aataaactga
acttaacact ggacttccac agactcctaa 1020cagtggagct tcagtttaaa
ctgaaagcca ttaatctgca gacagttcgt catcaagaac 1080tccctgactg
ttatgacttt actctgacta taacatttga caacaaggcc catagtggaa
1140gaattaaaat aagtttagat aatgacattt ccatcagaga atgtaaagac
tggcatgtat 1200ctggatcaat tcagaagaac actcattaca tgatgatctt
tgatgccttt gtcattctga 1260cttgcttggt ttcattaatc ctctgcatta
gatctgtgat tagaggactt cagcttcagc 1320aggtagggaa cgttgctttc
taggaatgct actgacattt tgattgacag agacattcac 1380tgtgcctccc
ctcttttccc taaaggagtt tgtcaatttt ttcctcctcc attataagaa
1440ggaagtttct gtttctgatc aaatggaatt tgtcaatgga tggtacatta
tgattattat 1500tagtgacata ttgacaatca ttggatcaat tctaaaaatg
gaaatccaag ctaaggtaat 1560ttttttccta atcatgctat tgttagtgtc
agatttgcac taatggtaat gtatttttcc 1620agaatgtaag aattttcaga
atgaattgtt tcttccaaac tgtatatcaa gtagacttga 1680aattggtaat
ggtaattttc ttaaatctag tcaggaggtc tcttaggcag agtttttcaa
1740agtgtgatcc acaaaccatt gcatcagaat cattgggtgc ctggtaaagt
gtaccatgtt 1800agacctactg aattcagact cttcggcggg gcctgtgaat
tcttacacac accaaaattc 1860atacacaacc aaggtaacta aggtaagagt
tttttttttt ttttaatctt acaagaaatg 1920ctcgaatctt taacaaaaat
gagtggggct ataggggaaa gtgaggtcaa ggcactatgg 1980tgtgcatgct
tgcatttgtt tcctccgtcc attcaaagtg agaatgctcc cattttctta
2040ctttaccatt gatgtgctac aagcttattt attttaagac taacctagcc
taaaaatcaa 2100ctgtccccac aaaataaaaa tcacattaaa aaaactaata
gtgttcagac taatcttgct 2160caaacttatg tttccctagt cttgatgcaa
ctgattgagt cacctgggga gttggttata 2220aacctgggca gagaccccaa
atgcaatggc tcagagaaga taggagctta tttctgtctt 2280atgcaatagt
cagaatgggt tttacagact ggtgagtagc tcaacatctc acagtcattc
2340aggcacccat gttcctccca ttttgtttct ctgccacccc ttaaggactt
gccctgactg 2400catgattatt gccgtgttgc ctcaaacagg ttgcagctta
tgggaagcaa aaacacggta 2460tggtagaagc tctcccatag actgatggct
tggctcaaga gtggccgact ttatttctgt 2520acatatccca ctggatagaa
tttagtcaat cctaactgca gagggagcca gggaacacag 2580cccaggcatg
tgcctaggaa ggggagaatg ggtttaggtt gacacttagc agctgccact
2640atatgtggct atagtatgta tcattggaat agatgtttaa ctttagggac
aaataaaaaa 2700ccaaaacaaa aaaaggagta aggggagaga tttgcagcaa
atctttattt ttaccaacct 2760caactatcat taatttcagt gaaccctaaa
tggtatccaa caaaatatct ttctagacca 2820ttcaccgtct ctgcctcata
gatgatcata tcatgttttc ttctcttctg aaacctctaa 2880tacccttgtc
ctatcctcat tctaagctga tgaccttact tcctatttca caaaaataat
2940agaaaaaaaa aaaaaaaa 2958402727DNAHomo sapiens 40atagcctttc
aaatttcggt taatggtaac tctcatcagt tactcaagcc aaaaatcttg 60gattcatcac
agactcctct ctttcactaa tctcttcttc cccacatcta atccaagagg
120aaatcctatt gttctacctt cattggccag gcactgtgac tcatataatc
ccagcacatt 180gggaggctga ggcgggagga tggcttgagc ccaggagttc
cagaccagcc tgggaaacat 240agtgaggccc catctctaca aaaaattaaa
aaaattagca gtcaaggtgg catgtgtctg 300taatccaagc tacttggggg
gctgaggtgg gaggatttct tgagcccggg aagtcgaggc 360tgcattgagc
cacggttgtg ccacggcact ccagcctggg tgacagagtg agacccctgt
420ttccaaaaaa aaaaaaaaaa aaaaagaata aaaatcacct ggtcaactcc
actattacca 480gcctggtcca agctactatc tctcatttat attattgcaa
tagcctcctc actcctccaa 540caacctgctg tcaaccacag cagccaacat
ctgatcatat cacttctgtt tgtggttctc 600aaatctcccc aattgagtta
cagtaaaaga caaacttggt gagtgccacc ttatctctat 660aactgtatac
cctttctatt gctcactcca gccagatgca atatccttgc caagcaccct
720cctgtctcag ggcctttgca cttgccagtc cctgtgcctg gaaggcttct
cccctagatt 780tttgcatgac ttctccctcc ctcccttcag atctttgctc
aaatgccttc tttttagtgt 840atgtaaaatg acaaacccat acccattcct
tatcccctcc tctgaatttt ctcttcagca 900attatcagca gcaagtgtcc
caaagtttct attaacttat ttctgttgtc tctttcttcc 960ctccactaga
atgtaagctt tatgagagca gagacttttg tttgttcact gctttatcct
1020tagcacctaa aacagtgcct tactcatagt tacctcaata tttattgcca
aatgaatttc 1080tgctttataa tctgattata tttttccact ctctcttaga
gtctaactag ttatgatgtc 1140tgtagcatac ttcttgggac ttctaccatg
cttgtgtggc ttggagtcat ccgatacctc 1200ggtttctttg caaagtacaa
cctcctcatt ttgacccttc aggcagcgct gcccaatgtc 1260atcaggttct
gctgctgtgc agctatgatt tacttaggtt actgcttctg tggatggatc
1320gtgctggggc cttaccatga caagtttcgt tctctgaaca tggtctctga
gtgccttttc 1380tctctgataa atggagatga tatgtttgcc acgtttgcaa
aaatgcagca aaaaagttac 1440ttagtctggc tgtttagtag aatttacctc
tactcattca tcagcctctt tatatatatg 1500attttaagtc ttttcattgc
actgatcact gatacatacg aaacaattaa gcaataccaa 1560caagatggct
tcccagagac tgaacttcgt acatttatat cagaatgcaa agatctaccc
1620aactctggaa aatacagatt agaagatgac cctccagtaa ctttattctg
ctgttgtaaa 1680aagtagctat caggtttatc tgtactttag aggaaaatat
aatgtgtagc tgagttggaa 1740cactgtggat attctgagat cagatgtagt
atgtttgaag actgttattt tgagctaatt 1800gagacctata attcaccaat
aactgtttat atttttaaaa gcaatattta atgtctttgc 1860aactttatgc
tgggattgtt tttaaaaaaa actttaatga ggaaagctat tggattatta
1920ttatttcttg tttattttgc catggcttta gaatgtattc tgtatgcctc
tcttttgctc 1980tgatactgtt gctcctgcta ttctgattgt gcagactgta
taattagtgg aaaacaatcc 2040ttggtctgac tgtgactttg gacaactcag
taaccctggc ttggaccact ctcaggagtc 2100catccttgag agagtgggtg
tagttaccat ttatacagta atcattgcat tttaaaatct 2160tctcttgaaa
ggaagaataa gagtgcacca gaataagagc gcaccagaat aagagcacac
2220cagctaacaa tgtgatacgg ccatatgtca cttaaggatg gagatatgtt
ctgagaaatg 2280tgtcattagg cgattttgtc attaaacatc atagcatgta
cttccacaaa cctagatggt 2340atagcctact acacacctag gctatttggt
atagcctgtt ggtcctgggg tacaaatctg 2400tacaacatgt tactgtattg
aatacagtag gcaattgtaa ctcaatggta agtatctaaa 2460catagaaaag
ggacagtaaa aatatggttt tataatcttc tgggaccacc attgtatatg
2520cggtacatca ttgaccaaaa catcgttatc cagcatatga ctgtatttgg
ttatgaaagc 2580caactgttac ttgattctgc ttttagttct taagaggatc
aggcttttaa atactcattt 2640acaagctttc tatcctcctt cagtgttaaa
gtagaaagta aaaagagtat cttatacatg 2700catgaaatta aagcatatac caaatgc
2727
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